Methods for producing highly phosphorylated lysosomal hydrolases

ABSTRACT

The present invention provides highly phosphorylated lysosomal hydrolases, methods of modifying lysosomal hydrolases with the lysosomal targeting pathway enzymes GlcNAc-phosphotransferase and/or phosphodiester α-GlcNAcase.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] This invention relates generally to enzymes involved in thelysosomal targeting pathway and particularly to isolated and purifiedGlcNAc-phosphotransferase and phosphodiester α-GlcNAcase, nucleic acidsencoding the enzymes, processes for production of recombinantGlcNAc-phosphotransferase and phosphodiester α-GlcNAcase, and the use ofthe enzymes for the preparation of highly phosphorylated lysosomalenzymes that are useful for the treatment of lysosomal storage diseases.

[0003] 2. Description of the Prior Art

Lysosomes and Lysosomal Storage Diseases

[0004] Lysosomes are organelles in eukaryotic cells that function in thedegradation of macromolecules into component parts that can be reused inbiosynthetic pathways or discharged by the cell as waste. Normally,these macromolecules are broken down by enzymes known as lysosomalenzymes or lysosomal hydrolases. However, when a lysosomal enzyme is notpresent in the lysosome or does not function properly, the enzymesspecific macromolecular substrate accumulates in the lysosome as“storage material” causing a variety of diseases, collectively known aslysosomal storage diseases.

[0005] Lysosomal storage diseases can cause chronic illness and death inhundreds of individuals each year. There are approximately 50 knownlysosomal storage diseases, e.g., Pompe Disease, Hurler Syndrome, FabryDisease, Maroteaux-Lamy Syndrome (mucopolysaccharidosis VI), MorquioSyndrome (mucopolysaccharidosis IV), Hunter Syndrome(mucopolysaccharidosis II), Farber Disease, Acid Lipase Deficiency,Krabbe Disease, and Sly Syndrome (mucopolysaccharidosis VII). In each ofthese diseases, lysosomes are unable to degrade a specific compound orgroup of compounds because the enzyme that catalyzes a specificdegradation reaction is missing from the lysosome, is present in lowconcentrations in the lysosome, or is present at sufficientconcentrations in the lysosome but is not functioning properly.

[0006] Lysosomal storage diseases have been studied extensively and theenzymes (or lack thereof) responsible for particular diseases have beenidentified. Most of the diseases are caused by a deficiency of theappropriate enzyme in the lysosome, often due to mutations or deletionsin the structural gene for the enzyme. For some lysosomal storagediseases, the enzyme deficiency is caused by the inability of the cellto target and transport the enzymes to the lysosome, e.g., I-celldisease and pseudo-Hurler polydystrophy.

[0007] Lysosomal Storage diseases have been studied extensively and theenzymes (or lack thereof) responsible for particular diseases have beenidentified (Scriver, Beaudet, Sly, and Vale, eds., The Metabolic Basisof Inherited Disease, 6th Edition, 1989, Lysosomal Enzymes, Part 11,Chapters 61-72, pp. 1565-1839). Within each disease, the severity andthe age at which the disease presents may be a function of the amount ofresidual lysosomal enzyme that exists in the patient.

Lysosomal Targeting Pathway

[0008] The lysosomal targeting pathways have been studied extensivelyand the process by which lysosomal enzymes are synthesized andtransported to the lysosome has been well described. Kornfeld, S.(1986). “Trafficking of lysosomal enzymes in normal and disease states.”Journal of Clinical Investigation 77: 1-6 and Kornfeld, S. (1990).“Lysosomal enzyme targeting.” Biochem. Soc. Trans. 18: 367-374.Generally, lysosomal enzymes are synthesized by membrane-bound polysomesin the rough endoplastic reticulum (“RER”) along with secretoryglycoproteins. In the RER, lysosomal enzymes acquire N-linkedoligosaccharides by the en-bloc transfer of a preformed oligosaccharidefrom dolichol phosphate containing 2 N-acetylglucosamine, 9-mannose and3-glucose. Glycosylated lysosomal enzymes are then transported to theGolgi apparatus along with secretory proteins. In the cis-Golgi orintermediate compartment lysosomal enzymes are specifically and uniquelymodified by the transfer of GlcNAc-phosphate to specific mannoses. In asecond step, the GlcNAc is removed thereby exposing the mannose6-phosphate (“M6P”) targeting determinant. The lysosomal enzymes withthe exposed M6P binds to M6P receptors in the trans-Golgi and istransported to the endosome and then to the lysosome. In the lysosome,the phosphates are rapidly removed by lysosomal phosphatases and themannoses are removed by lysosomal mannosidases (Einstein, R. and Gabel,C. A. (1991). “Cell- and ligand-specific deposphorylation of acidhydrolases: evidence that the mannose 6-phosphate is controlled bycompartmentalization.” Journal of Cell Biology 112: 81-94).

[0009] The synthesis of lysosomal enzymes having exposed M6P iscatalyzed by two different enzymes, both of which are essential if thesynthesis is to occur. The first enzyme is UDP-N-acetylglucosamine:lysosomal enzyme N-Acetylglucosamine-1-phosphotransferase(“GlcNAc-phosphotransferase”) (E.C. 2.7.8.17). GlcNAc-phosphotransferasecatalyzes the transfer of N-acetylglucosamine-1-phosphate fromUDP-GlcNAc to the 6 position of α1,2-linked mannoses on the lysosonialenzyme. The recognition and addition of N-acetylgluocosamine-1-phosphateto lysosomal hydrolases by GlcNAc-phosphotransferase is the critical anddetermining step in lysosomal targeting. The second step is catalyzed byN-acetylglucosamine-1-phosphodiester α-N-Acetylglucosaminidase(“phosphodiester α-GlcNAcase”) (E.C. 3.1.4.45). Phosphodiesterα-GlcNAcase catalyzes the removal of N-Acetylglucosamine from theGlcNAc-phosphate modified lysosomal enzyme to generate a terminal M6P onthe lysosomal enzyme. Preliminary studies of these enzymes have beenconducted. Bao et al., in The Journal of Biological Chemistry, Vol. 271,Number 49, Issue of Dec. 6, 1996, pp. 31437-31445, relates to a methodfor the purification of bovine UDP-N-acetylglucosamine: Lysosomal enzymeN-Acetylglucosamine-1-phosphotransferase and proposes a hypotheticalsubunit structure for the protein. Bao et al., in The Journal ofBiological Chemistry, Vol. 271, Number 49, Issue of Dec. 6, 1996, pp.31446-31451, relates to the enzymatic characterization andidentification of the catalytic subunit for bovineUDP-N-acetylglucosamine: Lysosomal enzymeN-Acetylglucosamine-1-phosphotransferase. Kornfeld et al., in TheJournal of Biological Chemistry, Vol. 273, Number 36, Issue of Sep. 4,1998, pp. 23203-23210, relates to the purification and multimericstructure of bovine N-Acetylglucosamine-1-phosphodiesterα-N-Acetylglucosaminidase. However, the proprietary monoclonalantibodies required to isolate these proteins have not been madeavailable to others and the protein sequences for the enzymes used inthese preliminary studies have not been disclosed.

[0010] Although the lysosomal targeting pathway is known and thenaturally occurring enzymes involved in the pathway have been partiallystudied, the enzymes responsible for adding M6P in the lysosomaltargeting pathway are difficult to isolate and purify and are poorlyunderstood. A better understanding of the lysosomal targeting pathwayenzymes and the molecular basis for their action is needed to assistwith the development of effective techniques for the utilization ofthese enzymes in methods for the treatment of lysosomal storagediseases, particularly in the area of targeted enzyme replacementtherapy.

Treatment of Lysosomal Storage Diseases

[0011] Lysosomal storage diseases caused by the lack of enzymes can intheory be treated using enzyme replacement therapy, i.e., byadministering isolated and purified enzymes to the patient to treat thedisease. However, to be effective, the lysosomal enzyme administeredmust be internalized by the cell and transported to the lysosome.Naturally occurring enzymes and their recombinant equivalents, however,have been of limited value in enzyme replacement therapy because thepurified or recombinant lysosomal enzymes do not contain adequateamounts of exposed M6P, or contain undesirable oligosaccharides whichmediates their destruction. Without sufficient M6P, the administeredlysosomal enzyme cannot efficiently bind to M6P receptors and betransported to the lysosome. For example, human acid α-glucosidasepurified from placenta contains oligomannose oligosaccharides which arenot phosphorylated (Mutsaers, J. H. G. M., Van Halbeek, H.,Vliegenthart, J. F. G., Tager, J. M., Reuser, A. J. J., Kroos, M., andGaljaard, H. (1987). “Determination of the structure of the carbohydratechains of acid α-glucosidase from human placenta.” Biochimica etBiophysica Acta 911: 244-251), and this glycoform of the enzyme is notefficiently internalized by cells (Reuser, A. J., Kroos, M. A., Ponne,N. J., Wolterman, R. A., Loonen, M. C., Busch, H. F., Visser, W. J., andBolhuis, P. A. (1984). “Uptake and stability of human and bovine acidalpha-glucosidase in cultured fibroblasts and skeletal muscle cells fromglycogenosis type II patients.” Experimental Cell Research 155:178-189). As a result of the inability to purify or synthesize lysosomalenzymes with the desired oligosaccharide structures, these enzymepreparations are inefficiently targeted to affected cells and are oflimited effectiveness in the treatment of these diseases. There exists,therefore, a need for enzymes that can be used in enzyme replacementtherapy procedures, particularly highly phosphorylated enzymes that willbe efficiently internalized by the cell and transported to the lysosome.

SUMMARY OF THE INVENTION

[0012] It is, therefore, an object of the present invention to providebiologically active GlcNAc-phosphotransferase and phosphodiesterα-GlcNAcase as isolated and purified polypeptides.

[0013] It is another object of the present invention to provide nucleicacid molecules encoding GlcNAc-phosphotransferase and phosphodiesterα-GlcNAcase.

[0014] It is another object of the present invention to provideexpression vectors having DNA that encodes GlcNAc-phosphotransferase andphosphodiester α-GlcNAcase.

[0015] It is a further object of the present invention to provide hostcells that have been transfected with expression vectors having DNA thatencodes GlcNAc-phosphotransferase or phosphodiester α-GlcNAcase.

[0016] It is another object of the present invention to provide methodsfor producing recombinant GlcNAc-phosphotransferase and recombinantphosphodiester α-GlcNAcase by culturing host cells that have beentransfected or transformed with expression vectors having DNA thatencodes GlcNAc-phosphotransferase or phosphodiester α-GlcNAcase.

[0017] It is another object of the present invention to provide isolatedand purified recombinant GlcNAc-phosphotransferase and recombinantphosphodiester α-GlcNAcase.

[0018] It is another object of the present invention to provide methodsfor the preparation of highly phosphorlyated lysosomal enzymes that areuseful for the treatment of lysosomal storage diseases.

[0019] It is a further object of the present invention to provide highlyphosphorlyated lysosomal hydrolases that are useful for the treatment oflysosomal storage diseases.

[0020] It is still another object of the present invention to providemethods for the treatment of lysosomal storage diseases.

[0021] It is still another object of the present invention to providemonoclonal antibodies to capable of selectively binding to bovineGlcNAc-phosphotransferase and to bovine phosphodiester α-GlcNAcase.

[0022] These and other objects are achieved by recovering isolated andpurified biologically active GlcNAc-phosphotransferase andphosphodiester α-GlcNAcase and using the enzymes to obtain nucleic acidmolecules that encode for the enzymes. The nucleic acid molecules codingfor either enzyme are incorporated into expression vectors that are usedto transfect host cells that express the enzyme. The expressed enzyme isrecovered and used to prepare highly phosphorylated lysosomal hydrolasesuseful for the treatment of lysosomal storage diseases. In particular,the enzymes are used to produce highly phosphorylated-lysosomalhydrolases that can be effectively used in enzyme replacement therapyprocedures.

[0023] Lysosomal hydrolases having high mannose structures are treatedwith GlcNAc-phosphotransferase and phosphodiester α-GlcNAcase resultingin the production of asparagine-linked oligosaccharides that are highlymodified with mannose 6-phosphate (“M6P”). The treated hydrolase bindsto M6P receptors on the cell membrane and is transported into the celland delivered to the lysosome where it can perform its normal or adesired function.

[0024] Other aspects and advantages of the present invention will becomeapparent from the following more detailed description of the inventiontaken in conjunction with the accompanying drawings.

BRIEF OF DESCRIPTION OF THE DRAWINGS

[0025]FIG. 1 shows a model of the subunit structure ofGlcNAc-phosphotransferase. The enzyme is a complex of six polypeptides.The α- and β-subunits are the product of a single gene. Followingtranslation, the α- and β-subunits are separated by proteolytic cleavagebetween Lys⁹²⁹ and Asp⁹³⁰. The α-subunit is a type II membraneglycoprotein with a single amino terminal membrane spanning domain. Theβ-subunit is a type I membrane spanning glycoprotein with a singlecarboxyl terminal membrane spanning domain. The γ-subunit is the productof a second gene. The γ-subunit is a soluble protein with a cleavedsignal peptide. The α-, β-, and γ-subunits are all tightly associated.

[0026]FIG. 2 shows a model of the subunit structure of phosphodiesterα-GlcNAcase. The enzyme is a tetramer composed of four identicalsubunits arranged as two non-covalently-associated dimers which arethemselves disulfide-linked. The single subunit is a type I membraneprotein containing a signal peptide, a pro region not present in themature enzyme and a single carboxyl terminal membrane spanning domain.

[0027]FIG. 3 shows a diagram of recombinant glycoprotein expression inCHO cells. In overexpressing CHO cells, the rh-GAA is processed alongthe pathways 1 and 2, depending on whether or not the enzyme is actedupon by GlcNAc-phosphotransferase (GnPT). Secreted GAA containspredominantly sialylated biantenniary complex-type glycans and is not asubstrate for GlcNAc-phosphotransferase. In the presence of the αl,2-mannosidase inhibitors, 1-deoxymannojirimycin or kifunensineconversion of MAN9 to MAN5 structures is blocked, resulting in secretionof GAA-bearing MAN7-9 structures which can be modified withGlcNAc-phosphotransferase and phosphodiester α-GlcNAcase (UCE)generating phosphorylated species (pathway 3).

[0028]FIG. 4 shows transient expression analysis of various plasmidconstucts of the α/β and γ subunits of human GlcNAc-phosphotransferase.Plasmids containing the α/β and/or the γ subunits were transfected into293T cells, the expressed protein was purified from the culture at 23,44.5 and 70 hours after transfection and relative amounts of expressionwere assessed by measuring phosphotransferase activity usingmethyl-α-D-mannoside and [β³²P] UDP-GlcNAc as substrates.

DETAILED DESCRIPTION OF THE INVENTION

[0029] The term “GlcNAc-phosphotransferase” as used herein refers toenzymes that are capable of catalyzing the transfer ofN-acetylglucosamine-1-phosphate from UDP-GlcNAc to the 6′ position ofα1,2-linked mannoses on lysosomal enzymes.

[0030] The term “phosphodiester α-GlcNAcase” as used herein refers toenzymes that are capable of catalyzing the removal ofN-Acetylglucosamine from GlcNAc-phosphate-mannose diester modifiedlysosomal enzymes to generate terminal M6P.

[0031] The terms “GlcNAc-phosphotransferase” and “phosphodiesterα-GlcNAcase” as used herein refer to enzymes obtained from anyeukaryotic species, particularly mammalian species such as bovine,porcine, murine, equine, and human, and from any source whether natural,synthetic, semi-synthetic, or recombinant. The terms encompassmembrane-bound enzymes and soluble or truncated enzymes having less thanthe complete amino acid sequence and biologically active variants andgene products.

[0032] The term “naturally occurring” as used herein means an endogenousor exogenous protein isolated and purified from animal tissue or cells.

[0033] The term “isolated and purified” as used herein means a proteinthat is essentially free of association with other proteins orpolypeptides, e.g., as a naturally occurring protein that has beenseparated from cellular and other contaminants by the use of antibodiesor other methods or as a purification product of a recombinant host cellculture.

[0034] The term “biologically active” as used herein means an enzyme orprotein having structural, regulatory, or biochemical functions of anaturally occurring molecule.

[0035] The term “nucleotide sequence” as used herein means apolynucleotide molecule in the form of a separate fragment or as acomponent of a larger nucleic acid construct that has been derived fromDNA or RNA isolated at least once in substantially pure form (i.e., freeof contaminating endogenous materials) and in a quantity orconcentration enabling identification, manipulation, and recovery of itscomponent nucleotide sequences by standard biochemical methods. Suchsequences are preferably provided in the form of an open reading frameuninterrupted by internal non-translated sequences, or introns that aretypically present in eukaryotic genes. Sequences of non-translated DNAmay be present 5′ or 3′ from an open reading frame where the same do notinterfere with manipulation or expression of the coding region.

[0036] The term “nucleic acid molecule” as used herein means RNA or DNA,including cDNA, single or double stranded, and linear or covalentlyclosed molecules. A nucleic acid molecule may also be genomic DNAcorresponding to the entire gene or a substantial portion therefor tofragments and derivatives thereof. The nucleotide sequence maycorrespond to the naturally occurring nucleotide sequence or may containsingle or multiple nucleotide substitutions, deletions and/or additionsincluding fragments thereof All such variations in the nucleic acidmolecule retain the ability to encode a biologically active enzyme whenexpressed in the appropriate host or an enzymatically active fragmentthereof. The nucleic acid molecule of the present invention may comprisesolely the nucleotide sequence encoding an enzyme or may be part of alarger nucleic acid molecule that extends to the gene for the enzyme.The non-enzyme encoding sequences in a larger nucleic acid molecule mayinclude vector, promoter, terminator, enhancer, replication, signalsequences, or non-coding regions of the gene.

[0037] The term “variant” as used herein means a polypeptidesubstantially homologous to a naturally occurring protein but which hasan amino acid sequence different from the naturally occurring protein(human, bovine, ovine, porcine, murine, equine, or other eukaryoticspecies) because of one or more deletions, insertions, derivations, orsubstitutions. The variant amino acid sequence preferably is at least50% identical to a naturally occurring amino acid sequence but is mostpreferably at least 70% identical. Variants may comprise conservativelysubstituted sequences wherein a given amino acid residue is replaced bya residue having similar physiochemical characteristics. Conservativesubstitutions are well known in the art and include substitution of onealiphatic residue for another, such as Ile, Val, Leu, or Ala for oneanother, or substitutions of one polar residue for another, such asbetween Lys and Arg; Glu and Asp; or Gln and Asn. Conventionalprocedures and methods can be used for making and using such variants.Other such conservative substitutions such as substitutions of entireregions having similar hydrophobicity characteristics are well known.Naturally occurring variants are also encompassed by the presentinvention. Examples of such variants are enzymes that result fromalternate mRNA splicing events or from proteolytic cleavage of theenzyme that leave the enzyme biologically active and capable ofperforming its catalytic function. Alternate splicing of mRNA may yielda truncated but biologically active protein such as a naturallyoccurring soluble form of the protein. Variations attributable toproteolysis include differences in the N- or C-termini upon expressionin different types of host cells due to proteolytic removal of one ormore terminal amino acids from the protein.

[0038] The term “substantially the same” as used herein means nucleicacid or amino acid sequences having sequence variations that do notmaterially affect the nature of the protein, i.e., the structure and/orbiological activity of the protein. With particular reference to nucleicacid sequences, the term “substantially the same” is intended to referto the coding region and to conserved sequences governing expression andrefers primarily to degenerate codons encoding the same amino acid oralternate codons encoding conservative substitute amino acids in theencoded polypeptide. With reference to amino acid sequences, the term“substantially the same” refers generally to conservative substitutionsand/or variations in regions of the polypeptide nor involved indetermination of structure or function.

[0039] The term “percent identity” as used herein means comparisonsamong amino acid sequences as defined in the UWGCG sequence analysisprogram available from the University of Wisconsin. (Devereaux et al.,Nucl. Acids Res. 12: 387-397 (1984)).

[0040] The term “highly phosphorylated lysosomal hydrolase” as used toherein means a level of phosphorylation on a purified lysosomalhydrolase which could not be obtained by only isolating the hydrolasefrom a natural source and without subsequent treatment with theGlcNAc-phosphotransferase and phosphodiester-α-GlcNAcase. In particular,“highly phosphorylated lysosomal hydrolase” means a lysosomal hydrolasethat contains from about 6% to about 100% bis-phosphorylatedoligosaccharides.

[0041] This invention is not limited to the particular methodology,protocols, cell lines, vectors, and reagents described because these mayvary. Further, the terminology used herein is for the purpose ofdescribing particular embodiments only and is not intended to limit thescope of the present invention. As used herein and in the appendedclaims, the singular forms “a,” “an,” and “the” include plural referenceunless the context clearly dictates otherwise, e.g., reference to “ahost cell”) includes a plurality of such host cells.

[0042] Because of the degeneracy of the genetic code, a multitude ofnucleotide sequences encoding GlcNAc-phosphotransferase, phosphodiesterα-GlcNAcase, or other sequences referred to herein may be produced. Someof these sequences will be highly homologous and some will be minimallyhomologous to the nucleotide sequences of any known and naturallyoccurring gene. The present invention contemplates each and everypossible variation of nucleotide sequence that could be made byselecting combinations based on possible codon choices. Thesecombinations are made in accordance with the standard triplet geneticcode as applied to the nucleotide sequence of naturally occurringGlcNAc-phosphotransferase or phosphodiester α-GlcNAcase, and all suchvariations are to be considered as being specifically disclosed.

[0043] Unless defined otherwise, all technical and scientific terms andany acronyms used herein have the same meanings as commonly understoodby one of ordinary skill in the art in the field of the invention.Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice of the present invention,the preferred methods, devices, and materials are described herein.

[0044] All patents and publications mentioned herein are incorporatedherein by reference to the extent allowed by law for the purpose ofdescribing and disclosing the proteins, enzymes, vectors, host cells,and methodologies reported therein that might be used with the presentinvention. However, nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

The Invention GlcNAc-phosphotransferase

[0045] In one aspect, the present invention provides isolated andpurified biologically active GlcNAc-phosphotransferase, nucleic acidmolecules encoding GlcNAc-phosphotransferase and its subunits,expression vectors having a DNA that encodes GlcNAc-phosphotransferase,host cells that have been transfected or transformed with expressionvectors having DNA that encodes GlcNAc-phosphotransferase, methods forproducing recombinant GlcNAc-phosphotransferase by culturing host cellsthat have been transfected or transformed with expression vectors havingDNA that encodes GlcNAc-phosphotransferase, isolated and purifiedrecombinant GlcNAc-phosphotransferase, and methods for usingGlcNAc-phosphotransferase for the preparation of highly phosphorylatedlysosomal enzymes that are useful for the treatment of lysosomal storagediseases.

[0046] To obtain isolated and purified GlcNAc-phosphotransferase and itssubunits and the nucleic acid molecules encoding the enzyme according tothe present invention, bovine GlcNAc phosphotransferase was obtained andanalyzed as follows. Splenocytes from mice immunized with a partiallypurified preparation of bovine GlcNAc-phosphotransferase were fused withmyeloma cells to generate a panel of hybridomas. Hybridomas secretingmonoclonal antibodies specific for GlcNAc-phosphotransferase wereidentified by immunocapture assay. In this assay, antibodies which couldcapture GlcNAc-phosphotransferase from a crude source were identified byassay of immunoprecipitates with a specific GlcNAc-phosphotransferaseenzymatic assay. Hybridomas were subcloned twice, antibody produced inascites culture, coupled to a solid support and evaluated forimmunoaffinity chromatography. Monoclonal PT18-Emphaze was found toallow a single step purification of GlcNAc-phosphotransferase tohomogeneity. Bao, et al., The Journal of Biological Chemistry, Vol. 271,Number 49, Issue of Dec. 6, 1996, pp. 31437-31445 relates to a methodfor the purification of bovine UDP-N-acetylglucosamine:Lysosomal-enzymeN-Acetylglucosamine-1-phosphotransferase and proposes a hypotheticalsubunit structure for the protein. Bao, et. al., The Journal ofBiological Chemistry, Vol. 271, Number 49, Issue of Dec. 6, 1996, pp.31446-31451. Using this technique, the enzyme was purified 488,000-foldin 29% yield. The eluted GlcNAc-phosphotransferase has a specificactivity of >10⁶, preferably >5×10⁶, more preferably >12×10⁶ pmol/mg andis apparently a homogenous, multi-subunit enzyme based on silver-stainedSDS-PAGE. The monoclonal antibody labeled PT18 was selected for use infurther experiments. A hybridoma secreting monoclonal antibody PT18 wasdeposited with the American Type Culture Collection, 10801 UniverisityBlvd., Manassas, Va. 20110 and assigned ATCC Accession No. ______.

[0047] GlcNAc-phosphotransferase was determined to be a complex of sixpolypeptides with α subunit structure α₂β₂γ₂. FIG. 1 shows a model ofthe subunit structure obtained from quantitative amino acid sequencing,immunoprecipitation with subunit-specific monoclonal antibodies,SDS-PAGE, and cDNA sequences. The evidence for the model is summarizedbelow. The molecular mass of the complex estimated by gel filtration is570,000 Daltons. The 166,000 Dalton α-subunit is found as adisulfide-linked homudimer. Likewise, the 51,000 Dalton γ-subunit isfound as a disulfide-linked homodimer. Because both the α- andγ-subunits are found in disulfide-linked homodimers, each molecule mustcontain at least one α- and one γ homodimer. Although the 56,000 Daltonβ-subunit is not found in a disulfide-linked homodimer, two independentlines of evidence strongly suggest each complex contains two β-subunitsas well. First, quantitative aminoterminal sequencing demonstrates a 1:1molar ratio between the β- and γ-subunits. Secondly, since the α- andβ-subunits are encoded by a single cDNA and divided by proteolyticprocessing, two β-subunits are produced for each α-subunit dimer. Thepredicted mass of the complex based on the composition α₂β₂γ₂ is 546,000Daltons (2×166,000+2×56,000+2×51,000) in excellent agreement with themass estimated by gel filtration.

[0048] GlcNAc-phosphotransferase was purified using an assay for thetransfer of GlcNAc-1-Phosphate to the synthetic acceptorα-methylmannoside. However, the natural acceptors forGlcNAc-phosphotransferase are the high mannose oligosaccharides oflysosomal hydrolases. To evaluate the ability of the purifiedGlcNAc-phosphotransferase to utilize glycoproteins as acceptors, thetransfer of GlcNAc-1-P to the lysosomal enzymes uteroferrin andcathepsin D, the nonlysosomal glycoprotein RNAse B, and the lysosomalhydrolase β-glucocerebrosidase (which is trafficked by a M6P independentpathway), were investigated. Both uteroferrin and cathepsin D areeffectively utilized as acceptors by purified GlcNAc-phosphotransferasewith K_(m)s below 20 μm. In contrast, neither RNAse B norβ-glucocerebrosidase is an effective acceptor.

[0049] The ineffectiveness of RNAse B, which contains a single highmannose oligosaccharide, as an acceptor is especially notable since theK_(m) was not reached at the solubility limit of the protein (at 600μm). This data clearly demonstrates the specific phosphorylation ofLysosomal hydrolases previously observed with crude preparations(Waheed, Pohlmann A., R., et al. (1982). “Deficiency ofUDP-N-acetylglucosamine:lysosomal enzymeN-Acetylglucosamine-1phosphotransferase in organs of I-Cell patients.”Biochemical and Biophysical Research Communications 105(3): 1052-10580is a property of the GlcNAc-phosphotransferase itself.

[0050] The α-subunit was identified as containing the UDP-GlcNAc bindingsite since this subunit was specifically photoaffinity-labeled with[β-³²P]-5-azido-UDP-Glc.

[0051] The amino-terminal and internal (tryptic) protein sequence datawas obtained for each subunit. N-terminal sequence was obtained fromeach subunit as follows. Individual subunits ofGlcNAc-phosphotransferase were resolved by polyacrylamide gelelectrophoresis in the presence of sodium dodecyl sulfate before andafter disulfide bond reduction. Subunits were then transferred to a PVDFmembrane by electroblotting, identified by Coomassie blue staining,excised, and subjected to N-terminal sequencing. To obtain internalsequence, GlcNAc-phosphotransferase was denatured, reduced, andalkylated, and individual subunits were resolved by gel filtrationchromatography. Isolated subunits were then digested with trypsin andthe tryptic peptides fractionated by reverse phase HPLC. Peaks whichappeared to contain only a single peptide were analyzed for purity byMALDI and subjected to N-terminal amino acid sequencing.

[0052] The amino acid sequence for the human α-subunit is shown in aminoacids 1-928 of SEQ ID NO:1; the human β-subunit in amino acids 1-328 ofSEQ ID NO:2; and the human γ-subunit in amino acids 25-305 of SEQ IDNO:3. The γ-subunit has a signal sequence shown in amino acids 1-24 ofSEQ ID NO:3.

[0053] Comparison with the databases using the blast algorithmsdemonstrate these proteins have not been previously described althoughseveral EST sequences of the corresponding cDNAs are present.

[0054] Using these peptide sequences and a combination of libraryscreening, RACE, PCR and Blast searching of expressed sequence tag(“EST”) files, full-length human cDNAs encoding each subunit were clonedand sequenced.

[0055] The nucleotide sequence for the human α/β-subunit precursor cDNAis shown in nucleotides 165-3932 of SEQ ID NO:4; the nucleotide sequencefor the α-subunit is shown in nucleotides 165-2948 of SEQ ID NO:4; thenucleotide sequence for the β-subunit is shown in nucleotides 2949-3932of SEQ ID NO:4; and the nucleotide sequence for the γ-subunit is shownin nucleotides 96-941 of SEQ ID NO:5. The nucleotide sequence for theγ-subunit signal peptide is shown in nucleotides 24-95 of SEQ ID NO:5.

[0056] For each subunit a N-terminal peptide and two internal peptidesequences have been identified in the respective cDNA sequence. Althoughthe protein sequence data is from the bovine protein and the cDNAsequences are human, the sequences are highly homologous (identities:α-subunit 43/50; β-subunit 64/64; γ-subunit 30/32), confirming thecloned cDNAs represent the human homologs of the bovineGlcNAc-phosphotransferase subunits. The α- and β-subunits were found tobe encoded by a single cDNA whose gene is on chromosome 12. Theγ-subunit is the product of a second gene located on chromosome 16. Theα/β-subunits precursor gene has been cloned and sequenced. The genespans ˜80 kb and contains 21 exons. The γ-subunit gene has also beenidentified in data reported from a genome sequencing effort. Theγ-subunit gene is arranged as 11 exons spanning 12 kb of genomic DNA.

[0057] Using the human cDNAs, the homologous murine cDNAs for the α-, β-and γ-subunits were isolated and sequenced using standard techniques.The murine α-β-subunit precursor cDNA is shown in SEQ ID NO:16. Thededuced amino acid sequence for the murine α-subunit is shown in SEQ IDNO:15 and the β-subunit in SEQ ID NO:8.

[0058] The mouse γ-subunit cDNA was isolated from a mouse liver libraryin λZap II using the γ-human γ-subunit cDNA as a probe. The humanγ-subunit cDNA was random hexamer-labeled with ³²P-dCTP and used toscreen a mouse liver cDNA library in λZap II. The probe hybridized tothree of 500,000 plaques screened. Each was subcloned to homogeneity,the insert excised, cloned into pUC19, and sequenced using standardmethods Sambrook, J., Fritsch E. F., et al. (1989). Molecular Cloning. ALaboratory Manual. Cold Spring Harbor, Cold Spring Harbor LaboratoryPress. The mouse γ-subunit cDNA sequence is shown in SEQ ID NO:10 andthe deduced amino acid sequence for the mouse γ-subunit is shown in SEQID NO:9.

[0059] Comparison of the deduced amino acid sequences of the human andmouse α-, β-, and γ-subunits demonstrates that the proteins are highlyhomologous with about an 80 percent identity.

[0060] To confirm that these enzymes were substantially the same betweenspecies, a partial homologous rat cDNA for the α- and β-subunits wasisolated and sequenced using standard techniques. The partial rat α- andβ-subunit cDNA is shown in SEQ ID NO:12. The deduced amino acid sequencecorresponding to the cDNA is shown in SEQ ID NO:11. Further, a partialhomologous Drosophila cDNA for the α- and β-subunits was isolated andsequenced using standard techniques. The partial Drosophila α- andβ-subunit cDNA is shown in SEQ ID NO:17. The deduced amino acid sequencecorresponding to the cDNA is shown in SEQ ID NO:13. Comparisons of thededuced amino acid sequences of the partial human, rat, and Drosophilaα- and β-subunits show that the proteins are highly homologous.

Phosphodiester α-GlcNAcase

[0061] In another aspect, the present invention provides isolated andpurified biologically active phosphodiester α-GlcNAcase, nucleic acidmolecules encoding phosphodiester α-GlcNAcase, expression vectors havinga DNA that encodes phosphodiester α-GlcNAcase, host cells that have beentransfected or transformed with expression vectors having DNA thatencodes phosphodiester α-GlcNAcase, methods for producing recombinantphosphodiester α-GlcNAcase by culturing host cells that have beentransfected or transformed with expression vectors having DNA thatencodes phosphodiester α-GlcNAcase, isolated and purified recombinantphosphodiester α-GlcNAcase, and methods for using phosphodiesterα-GlcNAcase for the preparation of highly phosphorylated lysosomalenzymes that are useful for the treatment of lysosomal storage diseases.

[0062] To obtain isolated and purified phosphodiester α-GlcNAcase andthe nucleic acid molecules encoding the enzyme according to the presentinvention, bovine phosphodiester α GlcNAcase was obtained and analyzedas follows. Mice were immunized with a partially purified preparation ofphosphodiester α-GlcNAcase and a functional screening strategy wasutilized to identify and isolate a monoclonal antibody specific forphosphodiester α-GlcNAcase. Immunogen was prepared by partiallypurifying phosphodiester α-GlcNAcase ˜6000-fold from a bovine pancreasmembrane pellet using chromatography on DEAE-Sepharose, iminodiaceticacid Sepharose, and Superose 6. Two BALBIc mice were each injectedintraperitoneally with 5 μg partially purified phosphodiesterα-GlcNAcase emulsified in Freunds complete adjuvant. On day 28, the micewere boosted intraperitoneally with 5 μg phosphodiester α-GlcNAcaseemulsified in Freunds incomplete adjuvant. On day 42 the mice were bledand an phosphodiester α-GlcNAcase specific immune response wasdocumented by “capture assay.” To perform the capture assay, serum (5μl) was incubated overnight with 1.2 units partially purifiedphosphodiester α-GlcNAcase. Mouse antibody was then captured on rabbitantimouse IgG bound to protein A-Ultralink™ resin. Following extensivewashing, bound phosphodiester α-GlcNAcase was determined in theUltralink pellet by assay of cleavage of [³H]-GlcNAc-1-phosphomannoseα-methyl.

[0063] Following a second intravenous boost with phosphodiesterα-GlcNAcase, the spleen was removed and splenocytes fused with SP2/0myeloma cells according to our modifications (Bag, M., Booth J. L., etal. (1996). “Bovine UDP-N-acetylglucosamine: lysosomal enzymeN-acetylglucosamine-1-phosphotransferase. I. Purification and subunitstructure.” Journal of Biological Chemistry 271: 31437-31445) ofstandard techniques; Harlow, E. and Lane, D. (1988). Antibodies: alaboratory manual, Cold Spring Harbor Laboratory). The fusion was platedin eight 96-well plates in media supplemented with recombinant humanIL-6 (Bazin, R. and Lemieux, R. (1989). “Increased proportion of B cellhybridomas secreting monoclonal antibodies of desired specificity incultures containing macrophage-derived hybridoma growth factor (IL-6).”Journal of Immunological Methods 116: 245-249) and grown untilhybridomas were just visible. Forty-eight pools of 16-wells wereconstructed and assayed for antiphosphodiester α-GlcNAcase activityusing the capture assay. Four pools were positive. Subpools of 4-wellswere then constructed from the wells present in the positive 16-wellpools. Three of the four 16-well pools contained a single 4-well poolwith anti-phosphodiester α-GlcNAcase activity. The 4 single wells makingup the 4-well pools were then assayed individually identifying the wellcontaining the anti-phosphodiester α-GlcNAcase secreting hybridomas.Using the capture assay, each hybridoma was subCloned twice and antibodyprepared by ascites culture. Monoclonals UC2 and UC3 were found to below affinity antibodies. UC1, a high affinity IgG monoclonal antibody,was prepared by ascites culture and immobilized on Emphaze forpurification of phosphodiester α-GlcNAcase. The monoclonal antibodylabeled UC1 was selected for use in further experiments. A hybridomasecreting monoclonal antibody UC1 was deposited with the American TypeCulture Collection, 10801 Univerisity Blvd., Manassas, Va. 20110 andassigned ATCC Accession No. ______.

[0064] To purify phosphodiester α-GlcNAcase, a solubilized membranefraction was prepared from bovine liver. Phosphodiester α-GlcNAcase wasabsorbed to monoclonal antibody UC1 coupled to Emphaze resin byincubation overnight with gentle rotation. The UC1-Emphaze was thenpacked in a column, washed sequentially with EDTA and NaHCO₃ at pH 7.0,then phosphodiester α(-GlcNAcase was eluted with NaHCO₃ at pH 10.Fractions containing phosphodiester α-GlcNAcase at specificactivities >50,000μ/mg were pooled and adjusted to pH 8.0 with ⅕thvolume of 1 M Tris HCl, pH 7.4. Following chromatography on UCI-Emphazethe phosphodiester α-GlcNAcase was purified 92,500-fold in 32% yield.

[0065] The phosphodiester α-GlcNAcase from UC1-Emphaze was concentratedand chromatographed on Superose 6. Phosphodiester α-GlcNAcase elutedearly in the chromatogram as a symmetric activity peak with a coincidentprotein peak. Following chromatography on Superose 6, the enzyme waspurified ˜715,000-fold in 24% yield. The purified enzyme catalyzed thecleavage of 472 μmols/hr/mg [³H]-GlcNAc-1-phosphomannose-α-methyl,corresponding to a specific activity of 472,000 units/mg.

[0066] The purified phosphodiester α-GlcNAcase was subjected to SDS-PAGEand protein was detected by silver staining (Blum, H., Beier H., et al.(1987). “Improved silver staining of plant proteins, RNA and DNA inpolyacrylamide gels.” Electrophoresis: 93-99). A diffuse band wasobserved with a molecular mass of approximately 70 kDa whose intensityvaries with the measured phosphodiester α-GlcNAcase activity. Thediffuse appearance of the band suggests the protein may be heavilyglycosylated. A faint band with a molecular mass of ˜150,000, which doesnot correlate with activity, was also present.

[0067] A model for the subunit structure of phosphodiester α-GlcNAcasewas determined by gel filtration chromatography and SDS-PAGE with andwithout disulfide bond reduction. The mass by gel filtration is about300,000. SDS-PAGE without disulfide bond reduction is ˜140,000.Following disulfide bond reduction, the apparent mass is 70,000.Together these data show phosphodiester α-GlcNAcase is a tetramercomposed of disulfide linked homodimers. FIG. 2 shows a model of thesubunit structure of phosphodiester α-GlcNAcase.

[0068] The amino terminal amino acid sequence of affinity purified,homogeneous bovine phosphodiester α-GlcNAcase was determined usingstandard methods (Matsudaira, P., Ed. (1993). A Practical Guide toProtein and Peptide Purification for Microsequencing. San Diego,Academic Press, Inc.). The pure enzyme was also subjected to trypsindigestion and HPLC to generate two internal tryptic peptides which weresequenced. The amino acid sequences of these three peptides are: Peptide1-Amino Terminal DXTRVHAGRLEHESWPPAAQTAGAHRPSVRTFV (SEQ ID NO:23);Peptide 2- Tryptic RDGTLVTGYLSEEEVLDTEN (SEQ ID NO:24): and Peptide 3-Tryptic GINLWEMAEFLLK (SEQ ID NO:25).

[0069] The protein, nucleotide, and EST data bases were searched forsequences that matched these peptide sequences and several human andmouse ESTs were found that had the sequence of the third peptide attheir amino termini. Three human infant brain EST clones and one mouseembryo clone were obtained from ATCC and sequenced. The three humanclones were all identical except for total length at their 3′ ends andvirtually identical to the mouse clone, except that the mouse ESTcontained a 102 bp region that was absent from all three human brainESTs. An EcoRI-HindIII fragment of about 700 bp was excised from thehuman cDNA clone (ATCC # 367524) and used to probe a human liver cDNAlibrary directionally cloned in TriplEx vector (Clontech). Of thepositive clones isolated from the library and converted to plasmids(pTriplEx), the largest (2200 bp) was represented by clone 6.5 which wasused for the rest of the analysis.

[0070] The cDNA clone has been completely sequenced on both strands andis a novel sequence that predicts a mature protein of about 50 kDa whichis in agreement with the size of the deglycosylated mature bovine liverphosphodiester α-GlcNAcase.

[0071] There is a unique BamHI site at base #512 and a unique Hind IDsite at base # 1581. All three bovine peptide sequences (peptides 1, 2,and 3) were found. Although the sequences of peptides 2 and 3 in thehuman are 100% identical to the bovine sequences, the amino-terminalpeptide in humans is only 67% identical to the bovine sequence. Thehuman liver clone contains the 102 base pair insert that has thecharacteristics of an alternatively spliced segment that was missing inthe human brain EST. The hydrophilicity plot indicates the presence of ahydrophobic membrane spanning region from amino acids 448 to 474 andanother hydrophobic region from amino acid 8 to 24 which fits the motiffor a signal sequence and there is a likely signal sequence cleavagesite between G24 and G25. There are six Asn-X-Ser/Thr potential N-linkedglycosylation sites, one of which is within the 102 bp insert. All ofthese sites are amino terminal of the putative trans-membrane region.These features indicate that the phosphodiester α-GlcNAcase is a type Imembrane spanning glycoprotein with the amino terminus in the lumen ofthe Golgi and the carboxyl terminus in the cytosol. This orientation isdifferent from that of other glycosyltransferases and glycosidasesinvolved in glycoprotein processing, which to date have been shown to betype II membrane spanning proteins.

[0072] The amino acid sequence for the phosphodiester α-GlcNAcasemonomer is shown in amino acids 50-515 of SEQ ID NO:6. The signalpeptide is shown in amino acids 1-24 of SEQ ID NO:6 and the pro segmentis shown in amino acids 2549 of SEQ ID NO:6. The human cDNA was clonedusing the techniques described above. The nucleotide sequence for themonomer that associates to form the phosphodiester α-GlcNAcase tetrameris shown in nucleotides 151-1548 of SEQ ID NO:7. The nucleotide sequencefor the signal sequence is shown in nucleotides 1-72 of SEQ ID NO:7. Thenucleotide sequence for the propeptide is shown in nucleotides 73-150 ofSEQ ID NO:7.

[0073] The murine cDNA for phosphodiester α-GlcNAcase is shown in SEQ IDNO:18. The deduced amino acid sequence for the murine phosphodiesterα-GlcNAcase is shown in SEQ ID NO:19. Comparison of the deduced aminoacid sequences of the human and mouse enzymes demonstrates that theproteins are highly homologous with about an 80 percent identity. Thisis especially true in the region of the active site where identityexceeds 90%. The murine gene for phosphodiester α-GlcNAcase is shown inSEQ ID NO:14.

[0074] The human phosphodiester α-GlcNAcase gene has been identified bydatabase searching. The sequence was determined during the sequencing ofclone 165E7 from chromosome 16.13.3, GenBank AC007011.1, gi4371266.Interestingly, the phosphodiester α-GlcNAcase gene was not identified bythe SCAN program used to annotate the sequence.

[0075] Because of the degeneracy of the genetic code, a DNA sequence mayvary from that shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:7 andstill encode a GlcNAc phosphotransferase and a phosphodiesterα-GlcNAcase enzyme having the amino acid sequence shown in SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:6. Such variant DNA sequencesmay result from silent mutations, e.g., occurring during PCRamplification, or may be the product of deliberate mutagenesis of anative sequence. The invention, therefore, provides equivalent isolatedDNA sequences encoding biologically active GlcNAc-phosphotransferase andphosphodiester α-GlcNAcase selected from: (a) the coding region of anative mammalian GlcNAc-phosphotransferase gene and phosphodiesterα-GlcNAcase gene; (b) cDNA comprising the nucleotide sequence presentedin SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:7; (c) DNA capable ofhybridization to the native mammalian GlcNAc-phosphotransferase gene andphosphodiester α-GlcNAcase gene under moderately stringent conditionsand which encodes biologically active GlcNAc-phosphotransferase andphosphodiester α-GlcNAcase; and (d) DNA which is degenerate as a resultof the genetic code to a DNA defined in (a), (b), or (c) and whichencodes biologically active GlcNAc-phosphotransferase and phosphodiesterα-GlcNAcase. GlcNAc-phosphotransferase and phosphodiester α-GlcNAcaseproteins encoded by such DNA equivalent sequences are encompassed by theinvention.

[0076] Those sequences which hybridize under stringent conditions andencode biologically functional GlcNAc-phosphotransferase andphosphodiester α-GlcNAcase are preferably at least 50-100% homologous,which includes 55, 60, 65, 70, 75, 75, 80, 85, 90, 95, 99% and allvalues and subranges therebetween. Homology may be determined with thesoftware UWCG as described above. Stringent hybridization conditions areknown in the art and are meant to include those conditions which allowhybridization to those sequences with a specific homology to the targetsequence. An example of such stringent conditions are hybridization at65° C. in a standard hybridization buffer and subsequent washing in 0.2×concentrate SSC and 0.1% SDS at 42-65° C., preferably 60° C. This andother hybridization conditions are disclosed in Sambrook, J., Fritsch E.F., et al. (1989). Molecular Cloning. A Laboratory Manual. Cold SpringHarbor, Cold Spring Harbor Laboratory Press. Alternatively, thetemperature for hybridization conditions may vary dependent on thepercent GC content and the length of the nucleotide sequence,concentration of salt in the hybridization buffer and thus thehybridization conditions may be calculated by means known in the art.

[0077] Recombinant Expression for GlcNAc-phosphotransferase andPhosphodiester α-GlcNAcase Isolated and purified recombinantGlcNAc-phosphotransferase and phosphodiester α-GlcNAcase enzymes areprovided according to the present invention by incorporating the DNAcorresponding to the desired protein into expression vectors andexpressing the DNA in a suitable host cell to produce the desiredprotein.

Expression Vectors

[0078] Recombinant expression vectors containing a nucleic acid sequenceencoding the enzymes can be prepared using well known techniques. Theexpression vectors include a DNA sequence operably linked to suitabletranscriptional or translational regulatory nucleotide sequences such asthose derived from mammalian, microbial, viral, or insect genes.Examples of regulatory sequences include transcriptional promoters,operators, enhancers, mRNA ribosomal binding sites, and appropriatesequences which control transcription and translation initiation andtermination. Nucleotide sequences are “operably linked” when theregulatory sequence functionally relates to the DNA sequence for theappropriate enzyme. Thus, a promoter nucleotide sequence is operablylinked to a GlcNAc-phosphotransferase or phosphodiester a GlcNAcase DNAsequence if the promoter nucleotide sequence controls the transcriptionof the appropriate DNA sequence.

[0079] The ability to replicate in the desired host cells, usuallyconferred by an origin of replication and a selection gene by whichtransformants are identified, may additionally be incorporated into theexpression vector.

[0080] In addition, sequences encoding appropriate signal peptides thatare not naturally associated with GlcNAc-phosphotransferase orphosphodiester α-GlcNAcase can be incorporated into expression vectors.For example, a DNA sequence for a signal peptide (secretory leader) maybe fused in-frame to the enzyme sequence so that the enzyme is initiallytranslated as a fusion protein comprising the signal peptide. A signalpeptide that is functional in the intended host cells enhancesextracellular secretion of the appropriate polypeptide. The signalpeptide may be cleaved from the polypeptide upon secretion of enzymefrom the cell.

Host Cells

[0081] Suitable host cells for expression of GlcNAc-phosphotransferaseand phosphodiester at α-GlcNAcase include prokaryotes, yeast, archae,and other eukaryotic cells. Appropriate cloning and expression vectorsfor use with bacterial, fungal, yeast, and mammalian cellular hosts arewell known in the art, e.g., Pouwels et al. Cloning Vectors: ALaboratory Manual, Elsevier, N.Y. (1985). The vector may be a plasmidvector, a single or double-stranded phage vector, or a single ordouble-stranded RNA or DNA viral vector. Such vectors may be introducedinto cells as polynucleotides, preferably DNA, by well known techniquesfor introducing DNA and RNA into cells. The vectors, in the case ofphage and viral vectors also may be and preferably are introduced intocells as packaged or encapsulated virus by well known techniques forinfection and transduction. Viral vectors may be replication competentor replication defective. In the latter case viral propagation generallywill occur only in complementing host cells. Cell-free translationsystems could also be employed to produce the enzymes using RNAs derivedfrom the present DNA constructs.

[0082] Prokaryotes useful as host cells in the present invention includegram negative or gram positive organisms such as E. coli or Bacilli. Ina prokaryotic host cell, a polypeptide may include a N-terminalmethionine residue to facilitate expression of the recombinantpolypeptide in the prokaryotic host cell. The N-terminal Met may becleaved from the expressed recombinant GlcNAc-phosphotransferase orphosphodiester α-GlcNAcase polypeptide. Promoter sequences commonly usedfor recombinant prokaryotic host cell expression vectors includeβ-lactamase and the lactose promoter system.

[0083] Expression vectors for use in prokaryotic host cells generallycomprise one or more phenotypic selectable marker genes. A phenotypicselectable marker gene is, for example, a gene encoding a protein thatconfers antibiotic resistance or that supplies an autotrophicrequirement. Examples of useful expression vectors for prokaryotic hostcells include those derived from commercially available plasmids such asthe cloning vector pBR322 (ATCC 37017). pBR322 contains genes forampicillin and tetracycline resistance and thus provides simple meansfor identifying transformed cells. To construct an expression vectorusing pBR322, an appropriate promoter and a DNA sequence are insertedinto the pBR322 vector.

[0084] Other commercially available vectors include, for example,pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1 (PromegaBiotec, Madison, Wis., USA).

[0085] Promoter sequences commonly used for recombinant prokaryotic hostcell expression vectors include β-lactamase (penicillinase), lactosepromoter system (Chang et al., Nature275:615, (1978); and Goeddel etal., Nature 281:544, (1979)), tryptophan (trp) promoter system (Goeddelet al., Nucl. Acids Res. 8:4057, (1980)), and tac promoter (Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,p. 412 (1982)).

[0086] Yeasts useful as host cells in the present invention includethose from the genus Saccharomyces, Pichia, K. Actinomycetes andKluyveromyces. Yeast vectors will often contain an origin of replicationsequence from a 2μ yeast plasmid, an autonomously replicating sequence(ARS), a promoter region, sequences for polyadenylation, sequences fortranscription termination, and a selectable marker gene. Suitablepromoter sequences for yeast vectors include, among others, promotersfor metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J.Biol. Chem. 255:2073, (1980)) or other glycolytic enzymes (Holland etal., Biochem. 17:4900, (1978)) such as enolase,glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvateedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. Other suitable vectors andpromoters for use in yeast expression are further described in Fleer etal., Gene, 107:285-195 (1991). Other suitable promoters and vectors foryeast and yeast transformation protocols are well known in the art.

[0087] Yeast transformation protocols are known to those of skill in theart. One such protocol is described by Hinnen et al., Proceedings of theNational Academy of Sciences USA, 75:1929 (1978). The Hinnen protocolselects for Trp.sup.+transformants in a selective medium, wherein theselective medium consists of 0.67% yeast nitrogen base, 0.5% casaminoacids, 2% glucose, 10 μg/ml adenine, and 20 μg/ml uracil.

[0088] Mammalian or insect host cell culture systems well known in theart could also be employed to express recombinantGlcNAc-phosphotransferase or phosphodiester α-GlcNAcase polypeptides,e.g., Baculovirus systems for production of heterologous proteins ininsect cells (Luckow and Summers, Bio/Technology 6:47 (1988)) or Chinesehamster ovary (CHO) cells for mammalian expression may be used.Transcriptional and translational control sequences for mammalian hostcell expression vectors may be excised from viral genomes. Commonly usedpromoter sequences and enhancer sequences are derived from Polyomavirus, Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus.DNA sequences derived from the SV40 viral genome may be used to provideother genetic elements for expression of a structural gene sequence in amammalian host cell, e.g., SV40 origin, early and late promoter,enhancer, splice, and polyadenylation sites. Viral early and latepromoters are particularly useful because both are easily obtained froma viral genome as a fragment which may also contain a viral origin ofreplication. Exemplary expression vectors for use in mammalian hostcells are well known in the art.

[0089] The enzymes of the present invention may, when beneficial, beexpressed as a fusion protein that has the enzyme attached to a fusionsegment. The fusion segment often aids in protein purification, e.g., bypermitting the fusion protein to be isolated and purified by affinitychromatography. Fusion proteins can be produced by culturing arecombinant cell transformed with a fusion nucleic acid sequence thatencodes a protein including the fusion segment attached to either thecarboxyl and/or amino terminal end of the enzyme. Preferred fusionsegments include, but are not limited to, glutathione-S-transferase,β-galactosidase, a poly-histidine segment capable of binding to adivalent metal ion, and maltose binding protein. In addition, the HPC-4epitope purification system may be employed to facilitate purificationof the enzymes of the present invention. The HPC-4 system is describedin U.S. Pat. No. 5,202,253, the relevant disclosure of which is hereinincorporated by reference.

Expression by Gene Activation Technology

[0090] In addition to expression strategies involving transfection of acloned cDNA sequence, the endogenous GlcNAc-phophotransfease andphosphodiester α-GlcNAcase genes can be expressed by altering thepromoter.

[0091] Methods of producing the enzymes of the present invention canalso be accomplished according to the methods of protein production asdescribed in U.S. Pat. No. 5,968,502, the relevant disclosure of whichis herein incorporated by reference, using the sequences forGlcNAc-phosphotransferase and phosphodiester α-GlcNAcase as describedherein.

Expression and Recovery

[0092] According to the present invention, isolated and purifiedGlcNAc-phosphotransferase or phosphodiester α-GlcNAcase enzymes may beproduced by the recombinant expression systems described above. Thcmethod comprises culturing a host cell transformed with an expressionvector comprising a DNA sequence that encodes the enzyme underconditions sufficient to promote expression of the enzyme. The enzyme isthen recovered from culture medium or cell extracts, depending upon theexpression system employed. As is known to the skilled artisan,procedures for purifying a recombinant protein will vary according tosuch factors as the type of host cells employed and whether or not therecombinant protein is secreted into the culture medium. When expressionsystems that secrete the recombinant protein are employed, the culturemedium first may be concentrated. Following the concentration step, theconcentrate can be applied to a purification matrix such as a gelfiltration medium. Alternatively, an anion exchange resin can beemployed, e.g., a matrix or substrate having pendant diethylaminoethyl(DEAE) groups. The matrices can be acrylamide, agarose, dextran,cellulose, or other types commonly employed in protein purification.Also, a cation exchange step can be employed. Suitable cation exchangersinclude various insoluble matrices comprising sulfopropyl orcarboxymethyl groups. Further, one or more reversed-phase highperformance liquid chromatography (RP-HPLC) steps employing hydrophobicRP-HPLC media (e.g., silica gel having pendant methyl or other aliphaticgroups) can be employed to further purify the enzyme. Some or all of theforegoing purification steps, in various combinations, are well known inthe art and can be employed to provide an isolated and purifiedrecombinant protein.

[0093] Recombinant protein produced in bacterial culture is usuallyisolated by initial disruption of the host cells, centrifugation,extraction from cell pellets if an insoluble polypeptide, or from thesupernatant fluid if a soluble polypeptide, followed by one or moreconcentration, salting-out, ion exchange, affinity purification, or sizeexclusion chromatography steps. Finally, RP-HPLC can be employed forfinal purification steps. Microbial cells can be disrupted by anyconvenient method, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents.

Preparation of Highly Phosphorylated Lysosomal Enzymes

[0094] In another aspect, the present invention provides highlyphosphorylated lysosomal hydrolases and methods for the preparation ofsuch hydrolases. The highly phosphorylated lysosomal hydrolases can beused in clinical applications for the treatment of lysosomal storagediseases.

[0095] The method comprises obtaining lysosomal hydrolases havingasparagine-linked oligosaccharides with high mannose structures andmodifying the α1,2-linked or other outer mannoses by the addition of M6Pin vitro to produce a hydrolase that can be used for the treatment oflysosomal storage diseases because it binds to cell membrane M6Preceptors and is readily taken into the cell and into the lysosome.Typically, the high mannose structures consist of from six to ninemolecules of mannose and two molecules of N-acetylglucosamine (GlcNAc).In the preferred embodiment, the high mannose structure is acharacteristic MAN7(D₂D₃) isomer structure consisting of seven moleculesof mannose and two molecules of N-acetylglucosamine (GlcNAc).

[0096] Highly phosphorylated Lysosomal hydrolases are produced bytreating the high mannose hydrolases with GlcNAc-phosphotransferasewhich catalyzes the transfer of N-acetylglucosamine-1-phosphate fromUDP-GlcNAc to the 6′ position of α1,2-linked or other outer mannoses onthe hydrolase. This GlcNAc-phosphotransferase modified hydrolase is thentreated with phosphodiester α-GlcNAcase which catalyzes the removal ofN-Acetylglucosamine to generate terminal M6P on the hydrolase.

[0097] In one embodiment of the invention, the GlcNAc-phosphotransferasetreated hydrolase may be isolated and stored without any subsequenttreatment. Subsequently, the GlcNAc-phosphotransferase treated hydrolasemay be modified further by treating the hydrolase with a phosphodiesterα-GlcNAcase.

[0098] Surprisingly, it has been found that the hydrolases containingM6P generated by this method are highly phosphorylated when compared tonaturally occurring or known recombinant hydrolases. The highlyphosphorylated lysosomal hydrolases of the present invention containfrom about 6% to about 100% bis-phosphorylated oligosaccharides comparedto less that about 5% bis-phosphorylated oligosaccharides on knownnaturally occurring or recombinant hydrolases.

[0099] These highly phosphorylated hydrolases have a higher affinity forthe M6P receptor and are therefore more efficiently taken into the cellby plasma membrane receptors. (Reuser, A. J., Kroos, M. A., Ponne, N.J., Wolterman, R. A., Loonen, M. C., Busch, H. F., Visser, W. J., andBolhuis, P. A. (1984). “Uptake and stability of human and bovine acidalpha-glucosidase in cultured fibroblasts and skeletal muscle cells fromglycogenosis type II patients.” Experimental Cell Research 155:178-189).

[0100] The high-affinity ligand for the cation-independent M6P receptoris an oligosaccharide containing two M6P groups (i.e., abis-phosphorylated oligosaccharide). Since a bisphosphorylatedoligosaccharides binds with an affinity 3500-fold higher than amonophosphorylated oligosaccharides, virtually all the high-affinitybinding of a lysosomal enzyme to the M6P receptor will result from thecontent of bis-phosphorylated oligosaccharides (Tong, P. Y., Gregory,W., and Kornfeld, S. (1989)). “Ligand interactions of thecation-independent mannose 6-phosphate receptor. The stoichiometry ofmannose 6-phosphate binding.” Journal of Biological Chemistry 264:7962-7969). It is therefore appropriate to use the content ofbis-phosphorylated oligosaccharides to compare the binding potential ofdifferent preparations of lysosomal enzymes.

[0101] The extent of mannose 6-phosphate modification of two differentlysosomal enzymes has been published. The oligosaccharide composition ofhuman α-galactosidase A secreted from Chinese hamster ovary cells hasbeen published (Matsuura, F., Ohta, M., Ioannou, Y. A., and Desnick, R.I. (1998). “Human alpha-galactosidase A: characterization of theN-linked oligosaccharides on the intracellular and secreted glycoformsoverexpressed by Chinese hamster ovary cells.” Glycobiology 8(4):329-39). Of all oligosaccharides on α-gal A released by hydrazinolysis,only 5.2% were bis-phosphorylated. Zhao et al. partially characterizedthe oligosaccharide structures on recombinant human α-iduronidasesecreted by CHO cells (Zhao, K. W., Faull, K. F., Kakkis, E. D., andNeufeld, E. F. (1997). “Carbohydrate structures of recombinant humanalpha-L-iduronidase secreted by Chinese hamster ovary cells.” J BiolChem 272(36): 22758-65) and demonstrated a minority of theoligosaccharides were bisphosphorylated. The qualitative techniquesutilized precluded the determination of the fraction of oligosaccharidesphosphorylated.

[0102] The production and secretion of human acid α-glucosidase by CHOcells has been reported (Van Hove, J. L., Yang, H. W., Wu, J. Y., Brady,R. O., and Chen, Y. T. (1996). “High level production of recombinanthuman lysosomal acid alpha-glucosidase in Chinese hamster ovary cellswhich targets to heart muscle and corrects glycogen accumulation infibroblasts from patients with Pompe disease.” Proceedings of theNational Academy of Sciences USA, 93(1): 6570). The carbohydratestructures of this preparation were not characterized in thispublication. However, this preparation was obtained and analyzed. Theresults, given in the examples below, showed that less than 1% of theoligosaccharides contained any M6P and bis-phosphorylatedoligosaccharides were not detectable. Together, these data show thatknown preparations of recombinant lysosomal enzymes contain no more than5.2% phosphorylated oligosaccharides. It appears that the preparation ofmore highly phosphorylated lysosomal enzymes is unlikely to be achievedwith known techniques. Naturally occurring human acid α-glucosidasepurified from human placenta contains very low levels of M6P (Mutsaers,I. H. G. M., Van Halbeek, H., Vliegenthart, J. F. G., Tager, J. M.,Reuser, A. J. J., Kroos, M., and Galjaard, H. (1987). “Determination ofthe structure of the carbohydrate chains of acid α-glucosidase fromhuman placenta.” Biochimica et Biophysica Acta 911: 244-251). Thearrangement of the phosphates as either bis- or monophosphorylatedoligosaccharides has not been determined, but less than 1% of theoligosaccharides contain any M6P.

[0103] The highly phosphorylated hydrolases of the present invention areuseful in enzyme replacement therapy procedures because they are morereadily taken into the cell and the lysosome. (Reuser, A. J., Kroos, M.A., Ponne, N. J., Wolterman, R. A., Loonen, M. C., Busch, H. F., Visser,W. J. and Bolhuis, P. A. (1984). “Uptake and stability of human andbovine acid alpha-glucosidase in cultured fibroblasts and skeletalmuscle cells from glycogenosis type II patients.” Experimental CellResearch 155: 178-189).

[0104] Any lysosomal enzyme that uses the M6P transport system can betreated according to the method of the present invention. Examplesinclude α-glucosidase (Pompe Disease), α-L-iduronidase (HurlerSyndrome), α-galactosidase A (Fabry Disease), arylsulfatase(Maroteaux-Lamy Syndrome), N-acetylgalactosamine-6-sulfatase orβ-galactosidase (Morquio Syndrome), iduronate 2-sulfatase (HunterSyndrome), ceramidase (Farber Disease), galactocerebrosidase (KrabbeDisease), β-glucuronidase (Sly Syndrome), Heparan N-sulfatase(Sanfilippo A), N-Acetyl-α-glucosaminidase (Sanfilippo B), AcetylCoA-α-glucosaminide N-acetyl transferase, N-acetyl-glucosamine-6sulfatase (Sanfilippo D), Galactose 6-sulfatase (Morquio A),Arylsulfatase A, B, and C (Multiple Sulfatase Deficiency), ArylsulfataseA Cerebroside (Metachromatic Leukodystrophy), Ganglioside (MucolipidosisIV), Acid β-galactosidase G_(M1) Galglioside (G_(M1) Gangliosidosis),Acid β-galactosidase (Galactosialidosis), Hexosaminidase A (Tay-Sachsand Variants), Hexosaminidase B (Sandhoff), α-fucosidase (Fucsidosis),α-N-Acetyl galactosaminidase (Schindler Disease), GlycoproteinNeuraminidase (Sialidosis), Aspartylglucosamine amidase(Aspartylglucosaminuria), Acid Lipase (Wolman Disease), Acid Ceramidase(Farber Lipogranulomatosis), Lysosomal Sphingomyelinase and otherSphingomyelinase (Nieman-Pick).

[0105] Methods for treating any particular lysosomal hydrolase with theenzymes of the present invention are within the skill of the artisan.Generally, the lysosomal hydrolase at a concentration of about 10 mg/mland GlcNAc-phosphotransferase at a concentration of about 100,000units/mL are incubated at about 37° C. for 2 hours in the presence of abuffer that maintains the pH at about 6-7 and any stabilizers orcoenzymes required to facilitate the reaction. Then, phosphodiesterα-GlcNAcase is added to the system to a concentration of about 1000units/mL and the system is allowed to incubate for about 2 more hours.The modified lysosomal enzyme having highly phosphorylatedoligosaccharides is then recovered by conventional means.

[0106] In a preferred embodiment, the lysosomal hydrolase at 10 mg/ml isincubated in 50 mm Tris-HCl, pH 6.7,5 mM MgCl₂, 5 mM MnCl₂, 2 mMUDP-GlcNAc with GlcNAc phosphotransferase at 100,000 units/mL at 37° C.for 2 hours. Phosphodiester α-GlcNAcase, 1000 units/mL, is then addedand the incubation continued for another 2 hours. The modified enzyme isthen repurified by chromatography on Q-Sepharose and step elution withNaCl.

Methods for Obtaining High Mannose Lysosomal Hydrolases

[0107] High mannose lysosomal hydrolases for treatment according to thepresent invention can be obtained from any convenient source, e.g., byisolating and purifying naturally occurring enzymes or by recombinanttechniques for the production of proteins. High mannose lysosomalhydrolases can be prepared by expressing the DNA encoding a particularhydrolase in any host cell system that generates a oligosaccharidemodified protein having high mannose structures, e.g., yeast cells,insect cells, other eukaryotic cells, transformed Chinese Hamster Ovary(CHO) host cells, or other mammalian cells.

[0108] In one embodiment, high mannose lysosomal hydrolases are producedusing mutant yeast that are capable of expressing peptides having highmannose structures. These yeast include the mutant S. cervesiae Δoch1,Δmnn1 (Nakanishi-Shindo, Y., Nakayama, K. I., Tanaka, A., Toda, Y. andJigami, Y. (1993). “Structure of the N-linked oligosaccharides that showthe complete loss of α-1,6-polymannose outer chain from ochl, ochl mnnl,and ochl mnn1 alg3 mutants of Saccharomyces cerevisiae.” Journal ofBiological Chemistry 268: 26338-26345).

[0109] Preferably, high mannose lysosomal hydrolases are produced usingover-expressing transformed insect, CHO, or other mammalian cells thatare cultured in the presence of certain inhibitors. Normally, cellsexpressing lysosomal hydrolases secrete acid α-glucosidase that containspredominantly sialylated biantenniary complex type glycans that do notserve as a substrate for GlcNAc-phosphotransferase and therefore cannotbe modified to use the M6P receptor.

[0110] According to the present invention, a new method has beendiscovered for manipulating transformed cells containing DNA thatexpresses a recombinant hydrolase so that the cells secrete high mannosehydrolases that can be modified according to the above method. In thismethod, transformed cells are cultured in the presence of αb1,2-mannosidase inhibitors and the high mannose recombinant hydrolasesare recovered from the culture medium. Inhibiting alpha 1,2-mannosidaseprevents the enzyme from trimming mannoses and forces the cells tosecrete glycoproteins having the high mannose structure. High mannosehydrolases are recovered from the culture medium using known techniquesand treated with GlcNAc-phosphotransferase and phosphodiesterα-GlcNAcase according to the method herein to produce hydrolases thathave M6P and can therefore bind to membrane M6P receptors and be takeninto the cell. Preferably, the cells are CHO cells and the hydrolasesare secreted with the MAN7(D₂D₃) structure. FIG. 3 shows the reactionscheme for this method.

[0111] In a preferred embodiment, recombinant human acid alphaglucosidase (“rh-GAA”) is prepared by culturing CHO cells secretingrh-GAA in Iscove's Media modified by the addition of an alpha1,2-mannosidase inhibitor. Immunoprecipitation of rh-GAA from the mediafollowed by digestion with either N-glycanase or endoglycosidase-Hdemonstrates that in the presence of the alpha 1,2-mannosidase inhibitorthe rh-GAA retains high mannose structures rather than the complexstructures found on a preparation secreted in the absence of theinhibitor. The secreted rh-GAA bearing high mannose structures is thenpurified to homogeneity, preferably by chromatography beginning with ionexchange chromatography on ConA-Sepharose, Phenyl-Sepharose and affinitychromatography on Sephadex G-100. The purified rh-GAA is then treated invitro with GlcNAc-phosphotransferase to convert specific mannoses toGlcNAc-phospho-mannose diesters. The GlcNAc-phosphomannose diesters arethen converted to M6P groups by treatment with phosphodiester aGlcNAcase. Experiments show that 74% of the rh-GAA oligosaccharides werephosphorylated, 62% being bis-phosphorylated, and 12%monophosphorylated. Since each molecule of rh-GAA contains 7 N-linkedoligosaccharides, 100% of the rh-GAA molecules are likely to contain themannose-phosphate modification.

[0112] Any alpha 1 ,2-mannosidase inhibitor can function in the presentinvention. Preferably, the inhibitor is selected from the groupconsisting of deoxymannojirimycin (dMM), kifunensine, D-Mannonolactamamidrazone, and N-butyl-deoxymannojirimycin. Most preferably theinhibitor is deoxymannojirimycin.

Treatment of Lysosomal Storage Diseases

[0113] In a further aspect, the present invention provides a method forthe treatment of lysosomal storage diseases by administering a diseasetreating amount of the highly phosphorylated lysosomal hydrolases of thepresent invention to a patient suffering from the correspondinglysosomal storage disease. While dosages may vary depending on thedisease and the patient, the enzyme is generally administered to thepatient in amounts of from about 0.1 to about 1000 milligrams per 50 kgof patient per month, preferably from about 1 to about 500 milligramsper 50 kg of patient per month. The highly phosphorylated enzymes of thepresent invention are more efficiently taken into the cell and thelysosome than the naturally occurring or less phosphorylated enzymes andare therefore effective for the treatment of the disease. Within eachdisease, the severity and the age at which the disease presents may be afunction of the amount of residual lysosomal enzyme that exists in thepatient. As such, the present method of treating lysosomal storagediseases includes providing the highly phosphorylated lysosomalhydrolases at any or all stages of disease progression.

[0114] The lysosomal enzyme is administered by any convenient means. Forexample, the enzyme can be administered in the form of a pharmaceuticalcomposition containing the enzyme and any pharmaceutically acceptablecarriers or by means of a delivery system such as a liposome or acontrolled release pharmaceutical composition. The term“pharmaceutically acceptable” refers to molecules and compositions thatare physiologically tolerable and do not typically produce an allergicor similar unwanted reaction such as gastric upset or dizziness whenadministered. Preferably, “pharmaceutically acceptable” means approvedby a regulatory agency of the Federal or a state government or listed inthe U.S. Pharmacopoeia or other generally recognized pharmacopoeia foruse in animals, preferably humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the compound isadministered. Such pharmaceutical carriers can be sterile liquids, suchas saline solutions, dextrose solutions, glycerol solutions, water andoils emulsions such as those made with oils of petroleum, animal,vegetable, or synthetic origin (peanut oil, soybean oil, mineral oil, orsesame oil). Water, saline solutions, dextrose solutions, and glycerolsolutions are preferably employed as carriers, particularly forinjectable solutions.

[0115] The enzyme or the composition can be administered by any standardtechnique compatible with enzymes or their compositions. For example,the enzyme or composition can be administered parenterally,transdermally, or transmucosally, e.g. orally or nasally. Preferably,the enzyme or composition is administered by intravenous injection.

[0116] The following Examples provide an illustration of embodiments ofthe invention and should not be construed to limit the scope of theinvention which is set forth in the appended claims. In the followingExamples, all methods described are conventional unless otherwisespecified.

EXAMPLES Materials and Methods

[0117] Lactating bovine udders were obtained from Mikkelson Beef, Inc.(Oklahoma City, Okla.). Ultrasphere ODS columns were obtained fromBeckman Instruments. Microsorb MV-NH₂ columns were obtained from RaininInstrument Co., Inc. (Woburn, Mass.). [γ³²P]ATP (7000 Ci/mmol; endlabeling grade), Na¹²⁵I, and Lubrol (C₁₆H₃₃(CH₂CH₂O)₂₃H) were obtainedfrom ICN (Costa Mesa, Calif.). Superose 6 (prep grade), DEAE-SepharoseFF, QAE-Sephadex A-25, molecular mass standards for SDS-PAGE,HiTrap-protein G columns, and Mono Q columns were obtained fromPharmacia Biotech Inc. 3M-Emphaze Biosupport Medium AB1, IODO GENiodination reagent, and the BCA protein assay reagent were obtained fromPierce. Glycerol, sucrose, α-methylmannoside, α-methylglucoside,reactive green 19-agarose, sodium deoxycholate, benzamidine, UDP-GlcNAc,phenylmethylsulfonyl fluoride, Tris, rabbit anti-mouse IgG, and mousemonoclonal antibody isotyping reagents were obtained from Sigma.

[0118] POROS 50 HQ was obtained from Perseptive Biosystems (Cambridge,Mass.). ProBlott polyvinylidene difluoride membranes were obtained fromApplied Biosystems Inc. (Foster City, Calif.). A Model QT12 rotarytumbler was obtained from LORTONE, Inc. (Seattle, Wash.). A mouseimmunoglobulin standard panel was obtained from Southern BiotechnologyAssociates, Inc. (Birmingham, Ala.). Recombinant interleukin-6, porcineuteroferrin, and monoclonal antibody BP95 were gifts from colleagues.Other chemicals were reagent grade or better and were from standardsuppliers.

Example 1 Preparation of Monoclonal Antibodies Specific for BovineGlcNAc-phosphotransferase

[0119] Bovine GlcNAc-phosphotransferase was partially purified 30,000fold as described (Bao, M., Booth J. L., et al. (1996). “BovineUDP-N-acetylglucosamine: Lysosomal enzymeN-acetylglucosamine-1-phosphotransferase. I. Purification and subunitstructure.” Journal of Biological Chemistry 271: 31437-31445) and usedto immunize mice. Spleens of immune mice were removed and spenocytesfused with SP2/0 myeloma cells according to Harlow (Harrow, E. and Lane,D. (1988). Antibodies: a laboratory manual, Cold Spring HarborLaboratory). The fusion was plated into 96 well plates and cultured inHAT media until hybridomas were visible.

[0120] Hybridomas secreting monoclonal antibodies capable of capturingGlcNAc-phosphotransferase from a crude sample were identified byincubation of hybridoma media (200 μl) with 200 units. Partiallypurified GlcNAc-phosphotransferase and capturing the resulting immunecomplex on rabbit anti-mouse IgG bound to protein A coupled toUltralink™ matrix. Immune complexes which contained monoclonalantibodies directed against GlcNAc-phosphotransferase were thenidentified by assay of the immune complex for GlcNAc-phosphotransferaseactivity. By this strategy, four monoclonals directed againstGlcNAc-phosphotransferase were identified in the fifth fusion screened.The hybridomas identified were subcloned twice using the same assay andascites was produced in BALBc mice according to standard techniques(Harlow, E. and Lane, D. (1988). Antibodies: a laboratory manual, ColdSpring Harbor Laboratory). The monoclonal antibody labeled PT18 wasselected for use in further experiments.

Example 2 Purification of Bovine GlcNAc-phosphotransferase

[0121] Lactating bovine mammary gland (6 kg) was collected at slaughterand immediately sliced into 10 cm thick slices and chilled in ice.Following homogenization in a Waring commercial blender, thepost-nuclear supernatant fraction was prepared by centrifugation.Membrane fragments were collected by high speed centrifugation (39,000×g, 45 minutes) and membrane proteins were solubilized in 4% Lubrol, 0.5%deoxycholate. GlcNAc-phosphotransferase was specifically adsorbed fromthe solubilized membrane fraction by incubation overnight with 10 ml ofmonoclonal antibody PT 18 coupled to Ultralink™ matrix (substitution 5mg/ml). The matrix was then collected by low speed centrifugation,washed with 0.025 M Tris-HCl, pH 7.4, 0.005 M MgCl₂, 0.3% Lubrol buffercontaining 1 M NaCl. The column was then washed with 2 column volumes of0.01 M Tris-HCl, pH 7.4, 0.005 M MgCl₂, 0.3% Lubrol buffer.GlcNAc-phosphotransferase was then eluted from the column with 0.10 MTris-HCl, pH 10.0, 0.005 M MgC₂, 0.3% Lubrol and neutralized with{fraction (1/10)}th volume of 1 M Tris-HCl, pH 6.0. Recovery istypically 20-50% of the GlcNAc-phosphotransferase activity present inthe homogenized tissue, and approximately 0.5 mg of enzyme is recoveredper 10 kg of tissue processed.

Example 3 Amino Acid Sequencing of Bovine GlcNAc-phosphotransferaseExample 3A Reduction, Alkylation and Separation of Individual Subunits

[0122] Bovine GlcNAc-phosphotransferase, 1.9 mg was desalted on a columnof Sephadex G-25 superfine equilibrated in 9% formic acid andlyophilized. The lyophilized protein was dissolved in 1 ml of 500 mMTris-HCl, pH 8.6, 6 M guanidine-HCl, 10 mM EDTA, 2 mM DTT degassed bybubbling N₂ gas through the solution and incubated at 37° C. for 1 hour.The solution was made 5 mM in iodoacetic acid and incubated at 37° C. inthe dark for a further 2½ hours. The solution was then made 15 mM inβ-mercaptoethanol and chromatographed on a column of Sephadex G-25superfine equilibrated in 9% formic acid. The void fraction was pooledand lyophilized. The individual subunits were resolved by chromatographyon a 1.0×30 cm column of Superose 12 equilibrated with 9% formic acid.

Example 3B Amino Terminal Sequencing of Individual Subunits

[0123] Bovine GlcNAc-phosphotransferase, 0.5 mg was equilibrated withsodium dodecyl sulfate, electrophoresed on a 6% polyacrylamide gel inthe presence of sodium dodecyl sulfate. The resolved subunits were thenelectro-transferred to a PVDF membrane and the protein bands detected bystaining with Coomassie Blue. The bands corresponding to the individualsubunits were then excised with a razor blade and subjected toamino-terminal sequencing in an Applied Biosystems Model 492 proteinsequencer. The amino terminal sequence of the α-subunit was Met Leu LeuLys Leu Leu Gin Arg Gin Arg Gin Thr Tyr (SEQ ID NO:26). The aminoterminal sequence of the β Subunit is Asp Thr Phe Ala Asp Ser Leu ArgTyr Val Asn Lys Ile Leu Asn Ser Lys Phe Gly Phe Thr Ser Arg Lys Val ProAla His (SEQ ID NO:27). The amino terminal sequence of the γ-subunit isAla Lys Met Lys Val Val Glu Glu Pro Asn Thr Phe Gly Leu Asn Asn Pro PheLeu Pro Gin (SEQ ID NO:28).

Example 3C Internal Amino Acid Sequence of the β- and γ-subunits

[0124] The resolved β- and γ-subunits from example 3B were treated withtrypsin at a 1/40 mass ratio overnight at 37° C. in 0.1 M Tris-HCl, pH8.0. The tryptic fragments were then resolved by reverse phasechromatography on a C18 column equilibrated with 0.1% trifluoroaceticacid and developed with a linear gradient in acetonitrile. Well resolvedpeaks were then subjected to amino terminal sequencing as described inexample 3B. The peptides sequenced from the βγ-subunit had the sequencesIle Leu Asn Ser Lys (SEQ ID NO:29), Thr Ser Phe His Lys (SEQ ID NO:30),Phe Gly Phe The Ser Arg (SEQ ID NO:31), and Ser Leu Val Thr Asn Cys LysPro Val Thr Asp Lys (SEQ ID NO:32). The peptide sequenced from theγ-subunit had the sequence Leu Ala His Val Ser Glu Pro Ser Thr Cys ValTyr (SEQ ID NO:33). A second peptide sequence from the γ-subunit wasobtained by chymotryptic digestion with the sequence Asn Asn Pro Phe LeuPro Gln Thr Ser Arg Leu Gin Pro (SEQ ID NO:34).

Example 3D Internal Amino Acid Sequence of the α-subunit

[0125] Internal peptide sequences of the α-subunit were obtained asfollows. Bovine GlcNAc phosphotransferase was reduced, alkylated,electrophoresed and transferred to PVDF as previously described. Theα-subunit band was excised and tryptic peptides generated by in situdigestion with trypsin, eluted with acetonitrile/trifluoroacetic acidand fractionated by reverse phase HPLC. Individual peaks were thenexamined by Matrix Associated Laser Desorption-Ionization-MassSpectroscopy (MALDI-MS) and peaks containing a single mass weresubjected to amino terminal sequencing as above. The peptide sequencesdetermined from the α-subunit are Val Pro Met Leu Val Leu Asp Xaa AlaXaa Pro Thr Xaa Val Xaa Leu Lys (SEQ ID NO:35) and Glu Leu Pro Ser LeuTyr Pro Ser Phe Leu Ser Ala Ser Asp Val Phe Asn Val Ala Lys Pro Lys (SEQID NO:36).

Example 4 Cloning the Human GlcNAc-phosphotransferase α/β-subunit cDNA

[0126] The amino-terminal protein sequence determined from the isolatedbovine β-subunit was used to search the Expressed Sequence Tag (EST)data base using the program tblastn. Altschul, S. F., Gish W., et al.(1990). “Basic Local Alignment Search Tool.” Journal of MolecularBiology 215: 403-410. This search identified a partial mouse cDNApreviously identified during a positional cloning strategy. Cordes, S.P. and Barsh, G. S. (1994). “The mouse segmentation gene kr encodes anovel basic domain-leucine zipper transcription factor.” Cell 79:1025-11034.

[0127] A forward PCR primer was designed based on the mouse sequence andused with an oligo dT reverse primer for RT-PCR amplification of a 1,848bp product using mouse liver poly A RNA as template. The PCR product wascloned and sequenced and proved to contain all the determined β-subunitsequences, demonstrating it encoded the murine β-subunit.

[0128] The human β-subunit cDNA was cloned by screening a size selectedhuman placental cDNA library (Fischman, K., Edman J. C., et al. (1990).“A murinefer testis-specific transcript (ferT encodes a truncated ferprotein.” Molecular and Cellular Biology 10: 146-153) obtained from ATCCwith the random hexamer labeled murine β-subunit cDNA under conditionsof reduced stringency (55° C., 2× SSC). The remaining portion of theα/β-subunit precursor cDNA was cloned by a combination of a walkingstrategy beginning with the portion of the cDNA encoding the humanβ-subunit and standard library screening strategies. Additionally, ESTdata base searches were used to identify clones containing portions ofthe human α/β cDNA, which were obtained from the correspondingrepositories and sequenced. Together these strategies allowed thedetermination of the full length human α/β-subunits precursor cDNAsequence. A clone containing this sequence was assembled using theappropriate fragments and cloned into pUC19. The 5597 bp sequence isgiven in Sequence NO:4 and contains DNA sequences predicted to encodeprotein sequences homologous to all of the amino terminal and internalpeptide sequences determined from the bovine α- and β-subunits.

Example 5 Cloning the Human GlcNAc-phosphotransferase γ-subunit cDNA

[0129] The γ-subunit amino terminal and tryptic peptide sequences wereused to search the Expressed Sequence Tag (EST) data base using theprogram tblastn. Altschul, S. F., Gish W., et al. (1990). “Basic LocalAlignment Search Tool.” Journal of Molecular Biology 215: 403-10. Threehuman EST sequences were identified which were highly homologous to thedetermined bovine protein sequences. cDNA clone 48250 from which ESTsequence 280314 was determined was obtained from Genome Systems andsequenced using standard techniques. This clone contained a 1191 bpinsert which contained all the determined protein sequences and appearedto contain a signal sequence 5′ of the determined amino terminalsequence. The clone however lacked an initiator methionine or any 5′non-coding sequence. The 5′ portion of the cDNA was obtained by PCR. thereverse primer 5′=GCGAAGATGAAGGTGGTGGAGGACC-3′ (SEQ ID NO:37) and a T7promoter primer were used in a reaction along with template DNA from ahuman brain cDNA library in pCMV-SPORT(GIBCO). A 654 bp product wasobtained, cloned in pCR2.1 and sequenced. The sequence demonstrated theamplified product contained 23 bp of 5′ non-coding sequence, theinitiator methionine and the signal peptide identified in EST 280314. Afull length cDNA for the γ-subunit (pBC36) was assembled by ligating a75 bp EcoRI-ApaI fragment from the cloned PCR product, an ApaI-NotIfragment from clone 48250 and EcoRI-NotI cut pcDNA3 (Invitrogen).

Example 6 Cloning the Human GlcNAc-phosphotransferase α/β-subunit Gene

[0130] Plasmid DNA was prepared from a human brain cDNA library (LifeTechnologies) according to the manufacturers protocol. This DNA was usedas template for PCR using primers with the sequences5′-TGCAGAGACAGACCTATACCTGCC-3′ (SEQ ID NO:38) and 5′ACTCACCTCTCCGAACTG-GAAAG-3′ (SEQ ID NO:39) using Taq DNA polymerase andbuffer A from Fischer Scientific using 35 cycles of 94° C. 1 minute, 55°C. 1 minute, and 79° C. 1 minute. A 106 bp product was obtained,purified by agarose gel electrophoresis, isolated by GeneClean (Bio101)and cloned into pCR2. DNA sequencing determined the resulting plasmidpAD39 contained a 106 bp insert which was excised by digestion withEcoRI and submitted to Genome Systems for screening of a human genomicBAC library. Four human BACs were identified and BAC #14951 wassequenced. For sequencing BAC #14951 was submitted to a colleague'slaboratory at the University of Oklahoma. The BAC was then fragmented bynebulization, and fragments cloned into pUC18 and shotgun sequenced.Contigs were generated by computer analysis and gaps closed by primerwalking strategies. The sequence of the BAC spans 177,364 bp. TheGlcNAc-phosphotransferase α/β-subunits precursor gene spans ˜80 kb andis arranged as 21 exons.

Example 7 Cloning the Human GlcNAc-phosphotransferase γ-subunit Gene

[0131] The human γ-subunit gene was identified by blastn searching ofthe NCBI High Throughput Genomic Sequence (HGTS) database with the fulllength human Subunit cDNA sequence. The search identified a cloneHS316G12(gi 4495019) derived from human chromosome 16 which containedthe human γ-subunit gene. The human GlcNAc-phosphotransferase γ-subunitgene spans about 12 kb and is arranged as 11 exons. Exons 1-3 and 4-11are separated by a large intron of about 9 kb.

Example 8 Preparation of Modified Expression Plasmid for the HumanGlcNAc-phosphotransferase α/β-subunits Precursor cDNA

[0132] An expression vector for the GlcNAc-phosphotransferase α/β cDNAwas constructed in pcDNA3.1(+) as follows. Two upstream ATG's in the5′-noncoding sequence of the human GlcNAc-phosphotransferase cDNA wereremoved and the Kozak sequence were modified as follows. Two fragmentsfrom pAD98, which was the human GlcNAc-phosphotransferase ct/p cDNAcloned into pcDNA3.1(+), were excised. A 1068 bp XhoI-PstI fragment anda 9746 bp NheI-XhoI fragment were ligated with oligonucleotides withsequences 5′-CTAGCCACCATGGGGTTCAAGCTCTTGCA-3′ (SEQ ID NO:40) and5′-AGAGCTTGAACCCCATGGTGG-3′ (SEQ ID NO:41) generating pAD105. The poly Asequence near the 3′ end of the cDNA clone was removed by ligating aNheI-BglII fragment from the cDNA with NheI-BamHI cut vector pcDNA3.1(+)generating pAD128.

Example 9 Preparation of an Expression Plasmids for the HumanGlcNAc-phosphotransferase α/β Subunits Precursor cDNA

[0133] DNA sequencing of pAD128 identified deletion of an A in an AAAAAsequence (positions 2761-2765 shown in SEQ ID NO:4) that disrupted thecoding sequence. Plasmid pAD130 was constructed in an attempt to correctthis by ligating a 5929 bp NheI-MfeI fragment and a 2736 bp NheI-AgeIfragment (both from pAD128 with a 515 bp MfeI-AgeI fragment derived frompAD124). Plasmid pAD130 was then grown and subsequent sequencing ofplasmid pAD130 demonstrated that the AAAAA sequence had reverted to AAAAagain indicating instability in the sequence at this point.

[0134] In order to eliminate this instability the first AAA (position2761-2763 shown in SEQ ID NO:4) that codes for lysine was changed to AAG(also coding for lysine) so that the unstable AAAAA sequence was changedto a stable AAGAA without altering the encoded amino acid. PlasmidpAD130 was corrected by removing a 214 bp MfeI-DraIII fragment andreplacing it with a fragment with the correct sequence. The correctMfeI-DraIII fragment was prepared by PCR using pAD130 as a template withforward primer 5′-GAAGACACAATTGGCATACTTCACTGATAGCAAGAATACTGGGAGGCAACTAAAAGATAC-3′ (SEQ ID NO:42) (oligo TTI 25 with desired AAGAAsequence as underlined) and reverse primer 5′-ACTGCATATCCTCAGAATGG-3′(SEQ ID NO:43) (oligo TTI 24). The PCR fragment was subCloned into theEcoRV site of pBluescript KS II(+) (Stratagene) generating pMK16. Theinsert was sequenced for confirmation and the 215 bp MfeI-DraIIIfragment was prepared. To avoid MfeI-DraIII sites on the vector pcDNA3.1 (+) (Invitrogen), the NheI-XbaI fragment was prepared from pAD130and subcloned into the XbaI site of pUC19 (Life Technologies) toconstruct pMK15. pMK15 was cleaved with MfeI and DraIII and the 6317 bpfragment was purified and ligated with the MfeI-DraIII fragment frompMK16 to form pMK19 containing the desired stable sequence in pUC19.

[0135] The corrected cDNA for the α/β subunit was excised from pMK19 asa KpnI-XbaI fragment and subcloned between the KpnI and XbaI sites ofpcDNA6/V5/His-A and designated pMK25. Plasmid pMK25 containing the cDNAas shown in SEQ ID NO:20 where the nucleotide sequence for the modifiedhuman α/β-subunit precursor cDNA is shown in nucleotides 1-3768. Thissequence corresponds to and is a modification of the nucleotide sequence165-3932 shown in SEQ ID NO:4.

Example 10 Construction of Expression Vectors for Soluble, HumanGlcNAc-phosphotransferase α/β Subunits Precursor cDNA

[0136] Plasmid pMK19 was digested with BglII (cutting at positions 255and 2703 shown in SEQ ID NO:20) and self-ligated to reduce the length ofthe cDNA to be amplified from approx. 3.5 kb to 1 kb so that the 5′ and3′ ends of the cDNA can be modified by PCR to remove the transmembranedomains of the α and β subunits of human GlcNAc-phosphotransferase andused to construct expression vectors to produce solubleGlcNAc-phosphotransferase. This plasmid was designated pMK21. Thestrategy is that the nucleotides encoding the first 44 amino acidscontaining the transmembrane domain of the α subunit (nucleotides 1-132of SEQ ID NO:20) are replaced with a HindIII site, and nucleotidesencoding the last 47 amino acids containing the transmembrane domain ofthe β subunit (nucleotides 3628-3768 of SEQ ID NO:21) are replaced witha stop codon and a XbaI site.

[0137] Plasmid pMK21 was used as a template for PCR with the followingprimers: A forward primer (5′-TGGTTCTGAAGCTTAGCCGAGATCAATACCATG-3′ (SEQID NO:44), oligo TTI 76) containing a HindIII site (underlined) and asequence complementary to nucleotides 133 to 151 of SEQ ID NO:20(italics), which will produce the 5′-end of a PCR fragment that removesthe coding sequence of the first 44 amino acids comprising the putativetransmembrane domain of the α subunit. A reverse primer(5′-TAGTACACTCTAGActactaCTTCAATTTGTCTCGATAAG-3′ (SEQ ID NO:45), oligoTTI 78) containing a XbaI site (underlined), two stop codons (lowercase) and a sequence complementary to nucleotides 3608 to 3627 of SEQ IDNO:21 (italics), which will produce the 3′-end of a PCR fragment thatremoves the coding sequence of the last 47 amino acids comprising theputative transmembrane domain of the β subunit and replaces it with twostop codons. The resulting PCR fragment was subcloned into the EcoRVsite of pBluescript KS II+(Stratagene). This plasmid, designated pMK42,was sequenced to ensure no errors were introduced by PCR. TheBglII-BglII fragment (positions 255-2703 shown in SEG ID NO:20) whichwas previously removed was subcloned back into the BglII site of pMK42.The orientation of this fragment was determined to be correct and thisplasmid was designated pMK49. Thus, plasmid pMK49 contained a cDNAcomprising a 5′ HindIII site and a 3′ XbaI site flanking a coding regionfor the human GlcNAc-phosphotransferase α/β subunits precursor cDNA withthe α subunit putative transmembrane domain deleted and the putativetransmembrane domain of the β subunit replaced with two stop codons(soluble α/β-cDNA).

[0138] This “soluble α/β-cDNA” can now be conveniently sub-cloned intovectors constructed to contain the HPC4 epitope (used for rapidpurification of the soluble enzyme) and different secretion signalpeptides. These pcDNA6/V5/His-A+tag) vectors were constructed asfollows:

[0139] Synthetic oligonucleotide cassettes containing a 5′-NheI site anda 3′-HindIII site flanking nucleotide sequences coding for differentsecretion signal peptides and the nucleotide sequence coding for theHPC4 epitope were inserted into plasmid pcDNA6/V5/His-A cut with NheIand HindIII. The following plasmids were prepared with the indicatedcassette:

[0140] 1. pMK45—mouse immunoglobulin Kappa chain signal peptide(sequence in italics) and HPC4 epitope (sequence underlined)CTAGCCGCCACC ATGGAGACAGACACACTC CTGCTATGGGTACTGCTGCTC (SEQ ID NO:46)    GGCGGTGGTACC TC TGTCT GTGTGAGGACGATACCCATGACGACGAG TGGGTTCC AGGT TCCACTGGTGA CGAAGATCAGGTAGATCCGCGGTT AATC ACCCAAGGTCCAAGGTGACCACTGCTTCTAGTCCAT CTAGGCGCCAATTAG GACGGTA CT GCCATTCGA

[0141] 1. pMK44—a transferrin signal peptide sequence (in italics) andHPC4 epitope (sequence underlined) CTAGCGGTACCATGAGATT AGCAGTAGGCGCC TTATTAG TATGCGC AGTACT C (SEQ ID NO:47)   CGCCATGGTACTCTAATCGTCATCCGCGGAATAATCATACGCGTCATGAG GGATTAT GTC TCGCAGAAGATCAGGTAGATCCGC GGTT AATCGACGGTA CCTTATACAGAGCGTCTTCTAG TCCATCTAGGCGCCAAT TAGCTGCCATTCGA

[0142] 1. pMK43—a transferrin secretion peptide sequence modified tosatisfy a Kozak's sequence(sequence in italics) and HPC4 epitope(sequence underlined), CTAGCCGCCACCATGGGATT AGCAGTAGGCGCCTT ATT AGTATGCGC AGT (SEQ ID NO:48)   CGCCGGTGGTACCCTAATCGTCATCCGCGGAATAATCATACGCGTCA ACT CGGATTAT GT CTCGCA GAAGATCAGGTAGATCCGC GGTTAATCGACG TGAGCCTAATACAGAGCGTCTT CTAGTCCATCTAGGCGCCAAT TAGCTGC GTA CATTCGA

[0143] The cDNA encoding “soluble α/β subunits” can be obtained as aHindIII-XbaI fragment from pMK49 and inserted into the plasmid pMK43 toform pMK50; pMK44 to form pMK51, and into pMK45 to form pMK52, plasmidscapable of encoding the α/β subunits of human GlcNAc-phosphotransferasewith putative transmembrane domains deleted, with different signalpeptides and all having the HPC4 epitope tag to facilitate purificationof the soluble, secreted enzyme.

Example 11 Construction of Expression Vectors for the HumanGlcNAc-phosphotransferase γ Subunit Precursor cDNA

[0144] The human GlcNAc-phosphotransferase 7-subunit precursor cDNA wasobtained from plasmid pAD133 in pAC5.1/V5-His by cutting with EcoRI.This cDNA was inserted into EcoRI digested pcDNA6/V5/His-A to formplasmid pMK17 containing cDNA as shown in SEQ ID NO:5. Plasmid pMK17 wasdigested with MluI (position 124-129 as shown in SEQ ID NO:5) and EcoRI(position 1103-1108 as shown in SEQ ID NO:5) and the 980 bp MluI-EcoRIfragment was then subcloned in pBluescriptKSII(+) with a syntheticdouble stranded cassette having an HindIII site and a MluI site flankinga nucleotide sequence including positions corresponding to 95-123 asshown in SEQ ID NO:5 thereby removing the nucleotide sequence encodingthe amino terminal, 24-amino acid signal peptide in plasmid pMK26.Plasmid pMK26 was sequenced to ensure its sequence. The correct cDNAfrom pMK26, which encodes amino acids for the humanGlcNAc-phosphotransferase γ subunit with the signal peptide removed, isthen excised from pMK26 by HindIII and EcoRI digestion and placed intoplasminds pMK43 to form pMK58; pMK44 to form pMK59, and into pMK45 toform pMK64, plasmids capable of encoding the γ subunit of humanGlcNAc-phosphotransferase with its signal peptide deleted, withdifferent signal peptides and all having the HPC4 epitope tag tofacilitate purification of the soluble, γ subunit.

[0145] To evaluate the behavior of α/β/γ secreted products, the α/βsubunit precursor and the γ subunit were co-expressed in thebi-cistronic vector pIRES (Clontech). This was accomplished bysubcloning α/β and γ cDNAs expressing the desired subunit with aselected signal peptide and the HPC4 Tag into NheI site (MCS-A) and XbaIsite (MCS-B) of pIRES, respectively.

Example 12 Transient Expression of the α/β and γ Subunits of HumanGlcNAc-phosphotransferase in 293T Cells

[0146] Plasmids were transfected into 293T cells using Fugene6 (Roche)according to manufacturer's instructions. Culture media was collected 23h, 44.5 h and 70 h after transfection. Aliquots of media containingexpressed protein was captured on anti-HPC4 monoclonal antibody (U.S.Pat. No. 5,202,253) conjugated with Ultralink beads (Pierce) byovernight incubation at 4° C. The beads were washed to remove unboundprotein and assayed directly for phosphotransferase activity asdescribed previously (REF).

[0147] Plasmids used for expression all containing a sequence encodingfor the HPC4 tag were as follows:

[0148] 1. pMK50—modified transferrin secretion peptide and α/β subunitin pcDNA6/V5/His4

[0149] 2. pMK51—transferrin secretion peptide and α/β subunit inpcDNA6/V5/His4

[0150] 3. pMK52—mouse immunoglobulin secretion peptide and α/β subunitin pcDNA6/V5/His4

[0151] 4. pMK75—modified transferrin secretion peptide and α/β subunitand modified transferrin secretion peptide and γ subunit in pIRES

[0152] 5. pMK81—transferrin secretion peptide and α/β subunit andtransferrin secretion peptide and γ subunit in pIRES

[0153] 6. pMK76—mouse immunoglobulin secretion peptide and α/β subunitand mouse immunoglobulin secretion peptide and γ in pIRES

[0154] The relative amounts of expression detected by assay forphosphotransferase using methyl-α-D-mannoside and UDP-[P-³²P]-GlcNAc assubstrates with cell transfected with pcDNA6/V5/His-4 as controls isshown in FIG. 4.

Example 13 Expression and Purification GlcNAc-phosphotransferase α/β/γ

[0155] For expression and purification of the enzyme, a modifiedexpression plasmid is constructed in a modified expression vectorderived from pEE14. The plasmid directs the synthesis of a solubleepitope tagged GlcNAc-phosphotransferase molecule. The α/β-subunitprecursor is modified as follows: The 5′ portion of the cDNA whichencodes the α-subunit cytoplasmic and transmembrane domain is deletedand replaced with nucleotides which encode the transferrin signalpeptide followed by amino acids which encode the epitope for monoclonalantibody HPC4. The 3′ portion of the cDNA is modified by the insertionof a stop codon before the β-subunit transmembrane segment. The vectorpEE14.1 (Lonza Biologics) is modified by the insertion of a 850 bpMluI-NcoI fragment containing a modified vascular endothelial growthfactor (VEGF) promoter at the unique MluI site in pEE14.1 This vectorencoding the modified GlcNAc-phosphotransferase α/β-subunit precursor isco-transfected with a wild type γ-subunit construct containing the VEGFpromoter in pEE14. 1 into CHO-K1 cells using Fugene6 and plated into 96well plates. Transfectants are selected in 25 μm methionine sulfoximineand the plasmid amplified by selection in 96 well plates with 50 μM, 100μM, 250 μM, and 500 μM methionine sulfoxirnine. Clones are picked intoduplicate 96 well plate and the highest expressing clones selected bydot blotting media and immuno-detection with monoclonal antibody HPC4.The highest expressing clone is expanded into cell factories. Therecombinant soluble epitope tagged GlcNAc-phosphotransferase is purifiedfrom the media by chromatography on monoclonal antibody HPC4 coupled toUltralink in the presence of 5 mM MgCl₂ and 1 mM CaCl₂. The solubleepitope tagged GlcNAc-phosphotransferase is eluted with 5 mM EGTA and 5mM MgCl₂.

Example 14 Preparation of Monoclonal Antibodies Specific for BovinePhosphodiester α-GlcNAcase

[0156] Murine monoclonal antibodies specific for bovine phosphodiesterα-GlcNAcase were generated by immunization of mice with a partiallypurified preparation of phosphodiester α-GlcNAcase. Spleens were thenremoved from immune mice and fused with SP2/O myeloma cells according tostandard techniques (Harrow, E. and Lane, D. (1988). Antibodies: alaboratory manual, Cold Spring Harbor Laboratory). Hybridomas wereplated in eight 96-well plates and grown until hybridomas were visible.Hybridomas secreting antibodies to phosphodiester α-GlcNAcase wereidentified measuring phosphodiester α-GlcNAcase activity inimmunoprecipitates prepared by incubation of a partially purifiedpreparation of phosphodiester α-GlcNAcase with pooled hybridomasupernatants. Pools from 16 and 4 wells were assayed followed byindividual wells. Monoclonal UC1 was identified by this protocol andcoupled to Ultralink™ for use in purification of phosphodiesterα-GlcNAcase.

Example 15 Purification of Bovine Phosphodiester α-GlcNAcase

[0157] Bovine calf liver (1 kg) was homogenized in 0.05 M Imidazole-HCl,pH 7.0, 0.15 M NaCl, 0.01 M EDTA and a washed post-nuclear supernatantwas prepared. Membranes were collected by centafugation at 30,000× g for30 minutes and washed three times with the above buffer. Membraneproteins were then solubilized in buffer containing 2% Triton X-100,0.05% deoxycholate and insoluble material removed by centrifugation, asbefore. The solubilized membrane fraction was incubated with 20 ml ofmonoclonal antibody UC1 coupled to Ultralink™ (substitution 5 mg/ml)with constant rotation for 16 hours at 4° C. The UC1-Ultralink™ wascollected by low speed centrifugation packed into a column and washedwith 0.025 M Tris-HCl, pH 7.4, 0.3% Lubrol, followed by two columnvolumes of 0.5 M NaHCO3, pH 8.0, 0.3% Lubrol. Phosphodiester α-GlcNAcasewas then eluted with 0.5 M NaHCO3, pH 10.0, 0.3% Lubrol and collected in1/10 volume of 1.0 M Tris-HCl, pH 5.5.

Example 16 Amino Acid Sequencing of Bovine Phosphodiester α-GlcNAcaseExample 16A Amino-terminal Sequence of Bovine Phosphodiester α-GlcNAcase

[0158] Bovine phosphodiester α-GlcNAcase was bound to a 0.25 ml columnof POROS HQ and step-eluted with buffer containing 0.5 M NaCl. Fractionscontaining phosphodiester α-GlcNAcase activity were identified byphosphodiester α-GlcNAcase assay, pooled and absorbed to a ProSorbSample Preparation Cartridge (Perkin Elmer) and subjected to amino acidsequencing in an Applied Biosystems Model 492 Protein Sequencer operatedaccording to the manufacturer's instructions. The sequenceAsp-Xaa-Thr-Arg-Val-His-Ala-Gly-Arg-Leu-Glu-His-Glu-Ser-Trp-Pro-Pro-Ala-Ala-Gln-Thr-Ala-Gly-Ala-His-Arg-Pro-Ser-Val-Arg-Thr-Phe-Valwas obtained.

Example 16B Internal Sequence of Bovine Phosphodiester α-GlcNAcase

[0159] Bovine liver phosphodiester α-GlcNAcase was concentrated to 10 μlin a Speed Vac, combined with 30 μl 0.1 M Tris-HCl, pH 7.4, 8 Mguanidine-HCl, and 24 μl 25 mM DTT and incubated at 50° C. for 1 hour.Iodoacetamide 2.4 μl 50 μM was then added and the incubation wascontinued for 1 hour. The reaction mixture was then desalted on a columnof Sephadex G25 superfine as described for GlcNAc-phosphotransferase anddigested with trypsin. The peptides were fractionated by HPLC andsequenced as described for GlcNAc-phosphotransferase. The sequencesdetermined are Arg Asp Gly Thr Leu Val Thr Gly Tyr Leu Ser Glu Glu GluVal Leu Asp Thr Glu Asn and Gly Ile Asn Leu Trp Glu Met Ala Glu Phe LeuLeu Lys.

Example 17 Cloning the Human Phosphodiester α-GlcNAcase cDNA

[0160] The phosphodiester α-GlcNAcase tryptic peptide sequences wereused to search the EST data bases as described forGlcNAc-phosphotransferase above. Three EST sequences were identifiedwhich contained the human phosphodiester α-GlcNAcase cDNA and clone ATCC#367524 was obtained and a ˜700 bp EcoRI-NotI fragment was excised fromthis clone and used to probe a human liver cDNA library in the vectorTriplEx. Several clones were identified and sequenced, one of which(clone 6.5) proved to contain a nearly full length cDNA for the humanphosphodiester α-GlcNAcase. The genomic clone described in Example 18demonstrated that clone 6.5 was missing only the initiator methionine.

Example 18 Cloning the Human Phosphodiester α-GlcNAcase Gene

[0161] The human phosphodiester α-GlcNAcase gene was identified bysearching the NCBI database nr with the human phosphodiester α-GlcNAcasecDNA using the program blastn. The genomic sequence was determinedduring the sequencing of a clone from chromosome 16p13.3 and depositedMar. 6, 1999 in GenBank as an unidentified sequence of 161264 bp withthe accession number AC007011. The gene spans about 12 kb of genomic DNAon chromosome 16.13 and is arranged in 11 exons.

Example 19 Construction of an Expression Vector for Human Phosphodiesteror α-GlcNAcase

[0162] An expression vector for human phosphodiester α-GlcNAcase wasprepared as follows: The 5′ end of the sequence of clone 6.5 wasmodified by PCR amplification of the 5′ end of the cDNA with a forwardprimer with the sequence 5′-GGAATTCCACCATGGCGACCTCCACGGGTCG-3′ (SEQ IDNO:49) and a reverse primer 5′-TGACCAGGGTCCCGTCGCG-3′ (SEQ ID NO:49).This served to add a consensus Kozak sequence and initiator methionineto the sequence of clone 6.5. The ˜500 bp PCR product was purified,digested with EcoRI and BamHI and ligated into pcDNA3. 1(−) which wassequenced. This construct was then digested with BamHI and HindIII andligated with a ˜1600 bp BamHI-HindIII fragment containing the 3′ portionof the cDNA from clone 6.5 generating the full length expressionplasmid.

Example 20 Host Cell Preparation for Human Phosphodiester α-GlcNAcase

[0163] Cos cells were grown in 60 mm plates in Dulbeccos minimalessential media (DMEM) at 37° C. in 5% CO₂ until they reached 50-80%confluence. The plates were then washed with OptiMEM I and the cellstransfected with the expression vector described in Example 19 usingLipofectamine Plus (GIBCO BRL Life Technologies) according to themanufacturers instructions. Cells were harvested at 48 hours, asolubilized membrane fraction prepared and assayed for phosphodiesterα-GlcNAcase activity.

Example 21 Expression and Purification of Soluble Recombinant HumanPhosphodiester α-GlcNAcase

[0164] For expression and purification of the enzyme, a modifiedexpression plasmid is constructed in a modified expression vectorderived from pEE14.1. The plasmid directs the synthesis of a solubleepitope tagged phosphodiester α-GlcNAcase molecule. The phosphodiesterα-GlcNAcase precursor is modified as follows: The 3′ portion of the cDNAwhich encodes the phosphodiester α-GlcNAcase transmembrane andcytoplasmic domains is deleted and replaced with nucleotides whichencode the epitope for monoclonal antibody HPC4 followed by a stopcodon. The vector pEE14.1 (Lonza Biologics) is modified by the insertionof a 850 bp MluI-NcoI fragment containing a modified vascularendothelial growth factor (VEGF) promoter at the unique MluI site inpEE14.1. This vector encoding the epitope tagged soluble phosphodiesterα-GlcNAcase precursor is transfected into CHO-K1 cells using Fugene6 andplated into 96 well plates. Transfectants are selected in 25 μmmethionine sulfoximine, and the plasmid amplified by selection in 96well plates with 50 μM, 100 μM, 250 μM, and 500 μM methioninesulfoximine. Clones are picked into duplicate 96 well plate and thehighest expressing clones selected by dot blotting media andimmuno-detection with monoclonal antibody HPC4. Media from clonesdemonstrating the highest level of epitope tag expression is assayed forphosphodiester α-GlcNAcase activity. The highest expressing clone isexpanded into cell factories. The recombinant soluble epitope taggedphosphodiester α-GlcNAcase is purified from the media by chromatographyon monoclonal antibody HPC4 coupled to Ultralink™ in the presence of 5mM MgCl₂ and 1 mM CaCl₂. The soluble epitope tagged phosphodiesterα-GlcNAcase is eluted with 5 mM EGTA and 5 mM MgCl₂.

Example 22 Construction of an Expression Vector for Soluble, HumanPhosphodiester α-GlcNAcase

[0165] For expression and purification of the enzyme, a modifiedexpression plasmid is constructed in a modified expression vectorderived from the pEE14.1 vector (Lonza Biologics). The plasmid directsthe synthesis of a soluble epitope tagged phosphodiester α-GlcNAcasemolecule. The phosphodiester α-GlcNAcase precursor is modified asfollows: The 3′ portion of the cDNA (1342-1548 of SEQ ID NO:7) whichencodes the phosphodiester α-GlcNAcase transmembrane and cytoplasmicdomains was deleted and replaced with nucleotide sequenceGAGGACCAGGTGGACCCCAGGCTGATCCAC GGCAAGGAT (SEQ ID NO:51) that encodes theepitope for monoclonal antibody HPC4 (EDQVDPRLIDGKD (SEQ ID NO:52))followed by a stop codon.

[0166] This expression vector was constructed by generating twointermediate plasmids and ligating a fragment from each into pEE14.1vector (Lonza Biologics) to yield the final expression vector. The firstintermediate plasmid designated pKB4 was constructed by ligating the1034 bp FseI>>Bsu36I fragment of phosphodiester α-GlcNAcase (lacking theC-terminal transmembrane and cytoplasmic domains) from clone 6.5, and aBsu36I-XbaI oligonucleotide fragment that contains the HPC4 epitope intoa modified pUC19 vector. The second intermediate plasmid designatedpKB5, was constructed by ligating a 850 bp MluI-NcoI fragment containinga modified vascular endothelial growth factor (VEGF) promoter frompcDNA4/HisMax (Invitrogen), a 256 bp BseI-FseI fragment encoding theN-terminus of human phosphodiester α-GlcNAcase from clone 6.5, and anoligonucleotide linker into a modified pUC19 vector. The finalexpression vector designated pKB6 was constructed by ligating theMluI-FseI fragment from pKB5, and the FseI-HindIII fragment from pKB4into a MluI/HindIII digested pEE14.1 vector. The plasmid pKB6 containsthe nucleotide sequence shown in SEQ ID NO:22.

Expression and Purification of Soluble Recombinant Human Phosphodiesterα-GlcNAcase

[0167] Approximately 10⁸ 293T cells were plated in a cell factory usingDulbecco's modified eagle's medium (DMEM) containing 10% fetal bovineserum in a humidified atmosphere at 37° C. with 5% CO2. These cells weretransfected with approximately 700 g of pKB6 using 2 ml of transfectionreagent Fugene-6 (Roche) for the transient expression of soluble humanphosphodiester α-GlcNAcase. After three days of culturing thetransfected cells, the medium containing soluble, epitope-tagged, humanphosphodiester α-GlcNAcase was collected and applied in the presence of1 mM CaCl2 to a column of monoclonal antibody HPC4 coupled to Ultralink(Pierce). Affinity purified, epitope-tagged, human phosphodiesterα-GlcNAcase (approximately 11 mg) was eluted with buffer containing 5 mMEDTA and stored at −20° C. in 50 mM Tris, 150 mM NaCl, 2 mM CaCl2, 50%glycerol, pH 7.2. The enzyme had a specific activity of 500,000 units/mgwith [³H]GlcNAc-phosphomannose-α-methyl as a substrate (Kornfeld R, etal., JBC 273:23203-23210).

Example 23 CHO Cells Expressing Recombinant Human Acid α-glucosidase

[0168] The human acid α-glucosidase cDNA was obtained from Dr. FrankMartinuk (Martiniuk, F., Mehler, M., Tzall, S., Meredith, G. andHirschhorn, R. (1990). “Sequence of the cDNA and 5′-flanking region forhuman acid alpha-glucosidase, detection of an intron in the 5′untranslated leader sequence, definition of 18-bp polymorphisms, anddifferences with previous cDNA and amino acid sequences.” DNA Cell Biol9(2): 85-94) and cloned into the expression vector pEE14.1. This vectorwas used to transfect CHO-K1 cells using Fugene6 and plated into 96 wellplates. Transfectants were selected in 25 μm methionine sulfoxirnine,and clones picked and plated into 96 well plates. The plasmid wasamplified by selection with 50 μM, 100 μM, 250μM, and 500 μM methioninesulfoximine. Clones were picked into duplicate 96 well plates and thehighest expressing clones selected by assay of the media for acidα-glucosidase activity and the cells for DNA content. The highestexpressing clone (Clone 3.49.13) based on acid α-glucosidase activity toDNA content ratio was then expanded into a cell factory. This clone wasincubated at 37° C. in 5% CO₂ and maintained in Glasgow MinimalEssential Media containing 20 mM TES, pH 7.2, 5% fetal bovine serum.

Example 24 Growth of CHO Cells Expressing Recombinant Human Acidα-glucosidase in the Presence of α-1,2 Mannosidase Inhibitors

[0169] CHO cells expressing human acid α-glucosidase were cultured inGlasgow Modified Minimal Essential Media containing 5% Fetal BovineSerum, 25 μM methionine sulfoximine, 20 mM TES, pH 7.2, and 7.5 mM1-deoxymannojirimycin-HCl. Alternatively, the cells can be cultured inthe above media containing 100 μg/mL 1-deoxymannojirimycin-HCl and 25μg/mL kifunensine.

Example 25 Isolation of Recombinant Human Acid α-glucosidase

[0170] Recombinant human acid α-glucosidase was purified from spenttissue culture media as follows: Media was concentrated 10 fold bytangential ultrafiltration with a 30,000 dalton cutoff membrane anddialyzed into 50 mM sodium phosphate, pH 6.5, and applied to a column ofConA Sepharose (Pharmacia). Following a wash with the same buffer toremove the unbound proteins, acid α-glucosidase was eluted with 1.0 Mα-methyl glucoside, pooled, concentrated and dialyzed as before. Theacid α-glucosidase was then applied to a column of Sephadex G-200equilibrated with 50 mM sodium phosphate, pH 6.5 and elutedisocratically with the same buffer.

Example 26 Treatment of Recombinant Human Acid α-glucosidase withGlcNAc-phosphotransferase and Phosphodiester α-GlcNAcase

[0171] Human acid α-glucosidase at 10 mg/ml was incubated in 50 mmTris-HCl, pH 6.7, 5 mM MgCl₂, 5 mM MnCl₂, 2 mM UDP-GlcNAc withGlcNAc-phosphotransferase at 100,000 u/mL at 37° C. for 2 hours.Phosphodiester α-GlcNAcase, 1000 u/mL was then added and the incubationcontinued for another 2 hours. The acid α-glucosidase was thenrepurified by chromatography on Q-Sepharose, and step elution with NaCl.

Example 27 Characterization of the Oligosaccharide Structures onModified Recombinant Human Acid α-glucosidase

[0172] Recombinant acid α-glucosidase treated or untreated withGlcNAc-phosphotransferase and phosphodiester α-GlcNAcase was digestedwith N-glycanase (New England Biolabs) or endomannosidase H (New EnglandBiolabs) according to the manufacturer's conditions. The releasedoligosaccharides were then labeled on the reducing terminus with2-aminobenzamide and fractionated by HPLC with fluorescent detectionaccording to the manufacturer's instructions (Oxford Glycosystems).Peaks were identified by comparison with standards chromatographed onthe same system, and confirmed by digestion with linkage specificglycosidases and/or mass determination by MALDI. The results are shownin Table 1. TABLE 1 Enzyme M6 M7 M8 M9 1P-Gn 2P-Gn 1M6P ComplexPreparation Rh-GAA 0 0 0 0 0 0 1 99 (Secreted) Rh-GAA 23 31 23 6 0 0 170 (dMM/ intracellular) Rh-GAA 6 11 7 2 12 62 0 0 (dMM/ intracellular)Ptase-treated

[0173] Referring to Table 1, the data (given in mole percent) show thatthe Lysosomal enzymes prepared using the GlcNAc-phosphotransferase andphosphodiester α-GlcNAcase of the present invention are highlyphosphorylated The data shows that the present invention produceslysosomal enzymes having about 5-10 M6P groups per enzyme compared toabout 0-2 for untreated enzymes and enzymes known in the art. Whencompared to naturally occurring or recombinant lysosomal enzymes, the invitro-modified preparation is very highly phosphorylated. In the mosthighly phosphorylated lysosomal enzyme known in the art, theα-galactosidase A described by Matsuura, F., Ohta, M., Ioannou, Y. A.and Desnick. R. J. (1998). “Human alpha-galactosidase A:characterization of the N-linked oligosaccharides on the intracellularand secreted glycoforms overexpressed by Chinese hamster ovary cells.”Glycobiology 8(4): 329-39, 5.2% of the oligosaccharides arebis-phosphorylated. In marked contrast, 62% of the oligosaccharides onthe in vitro-phosphorylated acid α-glucosidase, preparation describedhere contains bis-phosphorylated oligosaccharides. This represents abouta 12 fold increase. When the in vitro phosphorylated preparation ofrh-GAA shown in Table 1 is compared with GAA secreted from CHO cells bymethods known in the art, an even greater increase in phosphorylation isevident, about a 62 fold increase.

[0174] Thus, the in vitro-phosphorylated GAA is 12-62 fold morephosphorylated than any other described preparation of natural orrecombinant lysosomal enzyme. This difference has a major influence onthe rate and extent of internalization (Reuser, A. J., Kroos, M. A.,Ponne, N. J., Wolterman, R A., Loonen, M. C., Busch, H. F., Visser, W.J. and Bolhuis, P. A. (1984). “Uptake and stability of human and bovineacid alpha-glucosidase in cultured fibroblasts and skeletal muscle cellsfrom glycogenosis type II patients.” Experimental Cell Research 155:178-189).

Example 28 Comparison of Cell Uptake of Recombinant Human Acidα-glucosidase with or without Modification by GlcNAc-phosphotransferaseand Phosphodiester α-GlcNAcase

[0175] Human Pompe disease fibroblasts are obtained from ATCC andcultured in DMEM with 10% fetal bovine serum in 6 well plates andincubated at 37° C. in 5% CO₂. Recombinant human acid α-glucosidase withdifferent carbohydrate structures are compared for the rate and extentof internalization. Controls include each preparation incubated with 5mM mannose 6-phosphate and incubations without added recombinant humanacid α-glucosidase. The different preparations to be examined includeacid α-glucosidase secreted from CHO cells, acid α-glucosidase secretedfrom CHO cells in the presence of α1,2-mannosidase inhibitors, acidα-glucosidase secreted from CHO cells in the presence ofα1,2-mannosidase inhibitors treated with GlcNAc-phosphotransferase, andacid α-glucosidase secreted from CHO cells in the presence ofα1,2-mannosidase inhibitors treated with GlcNAc-phosphotransferase andphosphodiester α-GlcNAcase. Equal amounts of the four differentpreparations are added to each well and incubated at 37° C. for periodsvarying from 5 minutes to 4 hours. At the end of each incubation periodthe cell monolayers are washed with phosphate buffered saline containing5 mM mannose 6-phosphate and the monolayer solubilized in 1% TritonX-100 and assayed for internalized acid α-glucosidase by enzymaticassay.

[0176] Applicant and the assignee acknowledge their responsibility toreplace these cultures should they die before the end of the term of apatent issued hereon, 5 years after the last request for a culture, or30 years, whichever is the longer, and their responsibility to notifythe depository of the issuance of such a patent, at which time thedeposit will be made irrevocably available to the public. Until thattime the deposit will be made available to the Commissioner of Patentsunder the terms of 37 C.F.R 1.14 and 35 U.S.C. 112.

[0177] While the preferred embodiments are shown to illustrate theinvention, numerous changes to the materials and methods can be made bythose skilled in the art. All such changes are encompassed within thespirit of the invention as defined by the appended claims.

[0178] This application claims priority to U.S. Provisional applicationNo. 60/153,831 filed Sep. 14, 1999, and is incorporated herein byreference.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 52 <210> SEQ ID NO 1<211> LENGTH: 928 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400>SEQUENCE: 1 Met Leu Phe Lys Leu Leu Gln Arg Gln Thr Tyr Thr Cys Leu SerHis 1 5 10 15 Arg Tyr Gly Leu Tyr Val Cys Phe Leu Gly Val Val Val ThrIle Val 20 25 30 Ser Ala Phe Gln Phe Gly Glu Val Val Leu Glu Trp Ser ArgAsp Gln 35 40 45 Tyr His Val Leu Phe Asp Ser Tyr Arg Asp Asn Ile Ala GlyLys Ser 50 55 60 Phe Gln Asn Arg Leu Cys Leu Pro Met Pro Ile Asp Val ValTyr Thr 65 70 75 80 Trp Val Asn Gly Thr Asp Leu Glu Leu Leu Lys Glu LeuGln Gln Val 85 90 95 Arg Glu Gln Met Glu Glu Glu Gln Lys Ala Met Arg GluIle Leu Gly 100 105 110 Lys Asn Thr Thr Glu Pro Thr Lys Lys Ser Glu LysGln Leu Glu Cys 115 120 125 Leu Leu Thr His Cys Ile Lys Val Pro Met LeuVal Leu Asp Pro Ala 130 135 140 Leu Pro Ala Asn Ile Thr Leu Lys Asp ValPro Ser Leu Tyr Pro Ser 145 150 155 160 Phe His Ser Ala Ser Asp Ile PheAsn Val Ala Lys Pro Lys Asn Pro 165 170 175 Ser Thr Asn Val Ser Val ValVal Phe Asp Ser Thr Lys Asp Val Glu 180 185 190 Asp Ala His Ser Gly LeuLeu Lys Gly Asn Ser Arg Gln Thr Val Trp 195 200 205 Arg Gly Tyr Leu ThrThr Asp Lys Glu Val Pro Gly Leu Val Leu Met 210 215 220 Gln Asp Leu AlaPhe Leu Ser Gly Phe Pro Pro Thr Phe Lys Glu Thr 225 230 235 240 Asn GlnLeu Lys Thr Lys Leu Pro Glu Asn Leu Ser Ser Lys Val Lys 245 250 255 LeuLeu Gln Leu Tyr Ser Glu Ala Ser Val Ala Leu Leu Lys Leu Asn 260 265 270Asn Pro Lys Asp Phe Gln Glu Leu Asn Lys Gln Thr Lys Lys Asn Met 275 280285 Thr Ile Asp Gly Lys Glu Leu Thr Ile Ser Pro Ala Tyr Leu Leu Trp 290295 300 Asp Leu Ser Ala Ile Ser Gln Ser Lys Gln Asp Glu Asp Ile Ser Ala305 310 315 320 Ser Arg Phe Glu Asp Asn Glu Glu Leu Arg Tyr Ser Leu ArgSer Ile 325 330 335 Glu Arg His Ala Pro Trp Val Arg Asn Ile Phe Ile ValThr Asn Gly 340 345 350 Gln Ile Pro Ser Trp Leu Asn Leu Asp Asn Pro ArgVal Thr Ile Val 355 360 365 Thr His Gln Asp Val Phe Arg Asn Leu Ser HisLeu Pro Thr Phe Ser 370 375 380 Ser Pro Ala Ile Glu Ser His Ile His ArgIle Glu Gly Leu Ser Gln 385 390 395 400 Lys Phe Ile Tyr Leu Asn Asp AspVal Met Phe Gly Lys Asp Val Trp 405 410 415 Pro Asp Asp Phe Tyr Ser HisSer Lys Gly Gln Lys Val Tyr Leu Thr 420 425 430 Trp Pro Val Pro Asn CysAla Glu Gly Cys Pro Gly Ser Trp Ile Lys 435 440 445 Asp Gly Tyr Cys AspLys Ala Cys Asn Asn Ser Ala Cys Asp Trp Asp 450 455 460 Gly Gly Asp CysSer Gly Asn Ser Gly Gly Ser Arg Tyr Ile Ala Gly 465 470 475 480 Gly GlyGly Thr Gly Ser Ile Gly Val Gly His Pro Trp Gln Phe Gly 485 490 495 GlyGly Ile Asn Ser Val Ser Tyr Cys Asn Gln Gly Cys Ala Asn Ser 500 505 510Trp Leu Ala Asp Lys Phe Cys Asp Gln Ala Cys Asn Val Leu Ser Cys 515 520525 Gly Phe Asp Ala Gly Asp Cys Gly Gln Asp His Phe His Glu Leu Tyr 530535 540 Lys Val Ile Leu Leu Pro Asn Gln Thr His Tyr Ile Ile Pro Lys Gly545 550 555 560 Glu Cys Leu Pro Tyr Phe Ser Phe Ala Glu Val Ala Lys ArgGly Val 565 570 575 Glu Gly Ala Tyr Ser Asp Asn Pro Ile Ile Arg His AlaSer Ile Ala 580 585 590 Asn Lys Trp Lys Thr Ile His Leu Ile Met His SerGly Met Asn Ala 595 600 605 Thr Thr Ile His Phe Asn Leu Thr Phe Gln AsnThr Asn Asp Glu Glu 610 615 620 Phe Lys Met Gln Ile Thr Val Glu Val AspThr Arg Glu Gly Pro Lys 625 630 635 640 Leu Asn Ser Thr Ala Gln Lys GlyTyr Glu Asn Leu Val Ser Pro Ile 645 650 655 Thr Leu Leu Pro Glu Ala GluIle Leu Phe Glu Asp Ile Pro Lys Glu 660 665 670 Lys Arg Phe Pro Lys PheLys Arg His Asp Val Asn Ser Thr Arg Arg 675 680 685 Ala Gln Glu Glu ValLys Ile Pro Leu Val Asn Ile Ser Leu Leu Pro 690 695 700 Lys Asp Ala GlnLeu Ser Leu Asn Thr Leu Asp Leu Gln Leu Glu His 705 710 715 720 Gly AspIle Thr Leu Lys Gly Tyr Asn Leu Ser Lys Ser Ala Leu Leu 725 730 735 ArgSer Phe Leu Met Asn Ser Gln His Ala Lys Ile Lys Asn Gln Ala 740 745 750Ile Ile Thr Asp Glu Thr Asn Asp Ser Leu Val Ala Pro Gln Glu Lys 755 760765 Gln Val His Lys Ser Ile Leu Pro Asn Ser Leu Gly Val Ser Glu Arg 770775 780 Leu Gln Arg Leu Thr Phe Pro Ala Val Ser Val Lys Val Asn Gly His785 790 795 800 Asp Gln Gly Gln Asn Pro Pro Leu Asp Leu Glu Thr Thr AlaArg Phe 805 810 815 Arg Val Glu Thr His Thr Gln Lys Thr Ile Gly Gly AsnVal Thr Lys 820 825 830 Glu Lys Pro Pro Ser Leu Ile Val Pro Leu Glu SerGln Met Thr Lys 835 840 845 Glu Lys Lys Ile Thr Gly Lys Glu Lys Glu AsnSer Arg Met Glu Glu 850 855 860 Asn Ala Glu Asn His Ile Gly Val Thr GluVal Leu Leu Gly Arg Lys 865 870 875 880 Leu Gln His Tyr Thr Asp Ser TyrLeu Gly Phe Leu Pro Trp Glu Lys 885 890 895 Lys Lys Tyr Phe Gln Asp LeuLeu Asp Glu Glu Glu Ser Leu Lys Thr 900 905 910 Gln Leu Ala Tyr Phe ThrAsp Ser Lys Asn Thr Gly Arg Gln Leu Lys 915 920 925 <210> SEQ ID NO 2<211> LENGTH: 328 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400>SEQUENCE: 2 Asp Thr Phe Ala Asp Ser Leu Arg Tyr Val Asn Lys Ile Leu AsnSer 1 5 10 15 Lys Phe Gly Phe Thr Ser Arg Lys Val Pro Ala His Met ProHis Met 20 25 30 Ile Asp Arg Ile Val Met Gln Glu Leu Gln Asp Met Phe ProGlu Glu 35 40 45 Phe Asp Lys Thr Ser Phe His Lys Val Arg His Ser Glu AspMet Gln 50 55 60 Phe Ala Phe Ser Tyr Phe Tyr Tyr Leu Met Ser Ala Val GlnPro Leu 65 70 75 80 Asn Ile Ser Gln Val Phe Asp Glu Val Asp Thr Asp GlnSer Gly Val 85 90 95 Leu Ser Asp Arg Glu Ile Arg Thr Leu Ala Thr Arg IleHis Glu Leu 100 105 110 Pro Leu Ser Leu Gln Asp Leu Thr Gly Leu Glu HisMet Leu Ile Asn 115 120 125 Cys Ser Lys Met Leu Pro Ala Asp Ile Thr GlnLeu Asn Asn Ile Pro 130 135 140 Pro Thr Gln Glu Ser Tyr Tyr Asp Pro AsnLeu Pro Pro Val Thr Lys 145 150 155 160 Ser Leu Val Thr Asn Cys Lys ProVal Thr Asp Lys Ile His Lys Ala 165 170 175 Tyr Lys Asp Lys Asn Lys TyrArg Phe Glu Ile Met Gly Glu Glu Glu 180 185 190 Ile Ala Phe Lys Met IleArg Thr Asn Val Ser His Val Val Gly Gln 195 200 205 Leu Asp Asp Ile ArgLys Asn Pro Arg Lys Phe Val Cys Leu Asn Asp 210 215 220 Asn Ile Asp HisAsn His Lys Asp Ala Gln Thr Val Lys Ala Val Leu 225 230 235 240 Arg AspPhe Tyr Glu Ser Met Phe Pro Ile Pro Ser Gln Phe Glu Leu 245 250 255 ProArg Glu Tyr Arg Asn Arg Phe Leu His Met His Glu Leu Gln Glu 260 265 270Trp Arg Ala Tyr Arg Asp Lys Leu Lys Phe Trp Thr His Cys Val Leu 275 280285 Ala Thr Leu Ile Met Phe Thr Ile Phe Ser Phe Phe Ala Glu Gln Leu 290295 300 Ile Ala Leu Lys Arg Lys Ile Phe Pro Arg Arg Arg Ile His Lys Glu305 310 315 320 Ala Ser Pro Asn Arg Ile Arg Val 325 <210> SEQ ID NO 3<211> LENGTH: 305 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: SIGNAL <222> LOCATION: (1)..(24) <223> OTHERINFORMATION: <400> SEQUENCE: 3 Met Ala Ala Gly Leu Ala Arg Leu Leu LeuLeu Leu Gly Leu Ser Ala 1 5 10 15 Gly Gly Pro Ala Pro Ala Gly Ala AlaLys Met Lys Val Val Glu Glu 20 25 30 Pro Asn Ala Phe Gly Val Asn Asn ProPhe Leu Pro Gln Ala Ser Arg 35 40 45 Leu Gln Ala Lys Arg Asp Pro Ser ProVal Ser Gly Pro Val His Leu 50 55 60 Phe Arg Leu Ser Gly Lys Cys Phe SerLeu Val Glu Ser Thr Tyr Lys 65 70 75 80 Tyr Glu Phe Cys Pro Phe His AsnVal Thr Gln His Glu Gln Thr Phe 85 90 95 Arg Trp Asn Ala Tyr Ser Gly IleLeu Gly Ile Trp His Glu Trp Glu 100 105 110 Ile Ala Asn Asn Thr Phe ThrGly Met Trp Met Arg Asp Gly Asp Ala 115 120 125 Cys Arg Ser Arg Ser ArgGln Ser Lys Val Glu Leu Ala Cys Gly Lys 130 135 140 Ser Asn Arg Leu AlaHis Val Ser Glu Pro Ser Thr Cys Val Tyr Ala 145 150 155 160 Leu Thr PheGlu Thr Pro Leu Val Cys His Pro His Ala Leu Leu Val 165 170 175 Tyr ProThr Leu Pro Glu Ala Leu Gln Arg Gln Trp Asp Gln Val Glu 180 185 190 GlnAsp Leu Ala Asp Glu Leu Ile Thr Pro Gln Gly His Glu Lys Leu 195 200 205Leu Arg Thr Leu Phe Glu Asp Ala Gly Tyr Leu Lys Thr Pro Glu Glu 210 215220 Asn Glu Pro Thr Gln Leu Glu Gly Gly Pro Asp Ser Leu Gly Phe Glu 225230 235 240 Thr Leu Glu Asn Cys Arg Lys Ala His Lys Glu Leu Ser Lys GluIle 245 250 255 Lys Arg Leu Lys Gly Leu Leu Thr Gln His Gly Ile Pro TyrThr Arg 260 265 270 Pro Thr Glu Thr Ser Asn Leu Glu His Leu Gly His GluThr Pro Arg 275 280 285 Ala Lys Ser Pro Glu Gln Leu Arg Gly Asp Pro GlyLeu Arg Gly Ser 290 295 300 Leu 305 <210> SEQ ID NO 4 <211> LENGTH: 5597<212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 4cggagccgag cgggcgtccg tcgccggagc tgcaatgagc ggcgcccgga ggctgtgacc 60tgcgcgcggc ggcccgaccg gggcccctga atggcggctc gctgaggcgg cggcggcggc 120ggcggctcag gctcctcggg gcgtggcgtg gcggtgaagg ggtgatgctg ttcaagctcc 180tgcagagaca aacctatacc tgcctgtccc acaggtatgg gctctacgtg tgcttcttgg 240gcgtcgttgt caccatcgtc tccgccttcc agttcggaga ggtggttctg gaatggagcc 300gagatcaata ccatgttttg tttgattcct atagagacaa tattgctgga aagtcctttc 360agaatcggct ttgtctgccc atgccgattg acgttgttta cacctgggtg aatggcacag 420atcttgaact actgaaggaa ctacagcagg tcagagaaca gatggaggag gagcagaaag 480caatgagaga aatccttggg aaaaacacaa cggaacctac taagaagagt gagaagcagt 540tagagtgttt gctaacacac tgcattaagg tgccaatgct tgtactggac ccagccctgc 600cagccaacat caccctgaag gacgtgccat ctctttatcc ttcttttcat tctgccagtg 660acattttcaa tgttgcaaaa ccaaaaaacc cttctaccaa tgtctcagtt gttgtttttg 720acagtactaa ggatgttgaa gatgcccact ctggactgct taaaggaaat agcagacaga 780cagtatggag ggggtacttg acaacagata aagaagtccc tggattagtg ctaatgcaag 840atttggcttt cctgagtgga tttccaccaa cattcaagga aacaaatcaa ctaaaaacaa 900aattgccaga aaatctttcc tctaaagtca aactgttgca gttgtattca gaggccagtg 960tagcgcttct aaaactgaat aaccccaagg attttcaaga attgaataag caaactaaga 1020agaacatgac cattgatgga aaagaactga ccataagtcc tgcatattta ttatgggatc 1080tgagcgccat cagccagtct aagcaggatg aagacatctc tgccagtcgt tttgaagata 1140acgaagaact gaggtactca ttgcgatcta tcgagaggca tgcaccatgg gttcggaata 1200ttttcattgt caccaacggg cagattccat cctggctgaa ccttgacaat cctcgagtga 1260caatagtaac acaccaggat gtttttcgaa atttgagcca cttgcctacc tttagttcac 1320ctgctattga aagtcacatt catcgcatcg aagggctgtc ccagaagttt atttacctaa 1380atgatgatgt catgtttggg aaggatgtct ggccagatga tttttacagt cactccaaag 1440gccagaaggt ttatttgaca tggcctgtgc caaactgtgc cgagggctgc ccaggttcct 1500ggattaagga tggctattgt gacaaggctt gtaataattc agcctgcgat tgggatggtg 1560gggattgctc tggaaacagt ggagggagtc gctatattgc aggaggtgga ggtactggga 1620gtattggagt tggacacccc tggcagtttg gtggaggaat aaacagtgtc tcttactgta 1680atcagggatg tgcgaattcc tggctcgctg ataagttctg tgaccaagca tgcaatgtct 1740tgtcctgtgg gtttgatgct ggcgactgtg ggcaagatca ttttcatgaa ttgtataaag 1800tgatccttct cccaaaccag actcactata ttattccaaa aggtgaatgc ctgccttatt 1860tcagctttgc agaagtagcc aaaagaggag ttgaaggtgc ctatagtgac aatccaataa 1920ttcgacatgc ttctattgcc aacaagtgga aaaccatcca cctcataatg cacagtggaa 1980tgaatgccac cacaatacat tttaatctca cgtttcaaaa tacaaacgat gaagagttca 2040aaatgcagat aacagtggag gtggacacaa gggagggacc aaaactgaat tctacggccc 2100agaagggtta cgaaaattta gttagtccca taacacttct tccagaggcg gaaatccttt 2160ttgaggatat tcccaaagaa aaacgcttcc cgaagtttaa gagacatgat gttaactcaa 2220caaggagagc ccaggaagag gtgaaaattc ccctggtaaa tatttcactc cttccaaaag 2280acgcccagtt gagtctcaat accttggatt tgcaactgga acatggagac atcactttga 2340aaggatacaa tttgtccaag tcagccttgc tgagatcatt tctgatgaac tcacagcatg 2400ctaaaataaa aaatcaagct ataataacag atgaaacaaa tgacagtttg gtggctccac 2460aggaaaaaca ggttcataaa agcatcttgc caaacagctt aggagtgtct gaaagattgc 2520agaggttgac ttttcctgca gtgagtgtaa aagtgaatgg tcatgaccag ggtcagaatc 2580cacccctgga cttggagacc acagcaagat ttagagtgga aactcacacc caaaaaacca 2640taggcggaaa tgtgacaaaa gaaaagcccc catctctgat tgttccactg gaaagccaga 2700tgacaaaaga aaagaaaatc acagggaaag aaaaagagaa cagtagaatg gaggaaaatg 2760ctgaaaatca cataggcgtt actgaagtgt tacttggaag aaagctgcag cattacacag 2820atagttactt gggctttttg ccatgggaga aaaaaaagta tttccaagat cttctcgacg 2880aagaagagtc attgaagaca caattggcat acttcactga tagcaaaaat actgggaggc 2940aactaaaaga tacatttgca gattccctca gatatgtaaa taaaattcta aatagcaagt 3000ttggattcac atcgcggaaa gtccctgctc acatgcctca catgattgac cggattgtta 3060tgcaagaact gcaagatatg ttccctgaag aatttgacaa gacgtcattt cacaaagtgc 3120gccattctga ggatatgcag tttgccttct cttattttta ttatctcatg agtgcagtgc 3180agccactgaa tatatctcaa gtctttgatg aagttgatac agatcaatct ggtgtcttgt 3240ctgacagaga aatccgaaca ctggctacca gaattcacga actgccgtta agtttgcagg 3300atttgacagg tctggaacac atgctaataa attgctcaaa aatgcttcct gctgatatca 3360cgcagctaaa taatattcca ccaactcagg aatcctacta tgatcccaac ctgccaccgg 3420tcactaaaag tctagtaaca aactgtaaac cagtaactga caaaatccac aaagcatata 3480aggacaaaaa caaatatagg tttgaaatca tgggagaaga agaaatcgct tttaaaatga 3540ttcgtaccaa cgtttctcat gtggttggcc agttggatga cataagaaaa aaccctagga 3600agtttgtttg cctgaatgac aacattgacc acaatcataa agatgctcag acagtgaagg 3660ctgttctcag ggacttctat gaatccatgt tccccatacc ttcccaattt gaactgccaa 3720gagagtatcg aaaccgtttc cttcatatgc atgagctgca ggaatggagg gcttatcgag 3780acaaattgaa gttttggacc cattgtgtac tagcaacatt gattatgttt actatattct 3840cattttttgc tgagcagtta attgcactta agcggaagat atttcccaga aggaggatac 3900acaaagaagc tagtcccaat cgaatcagag tatagaagat cttcatttga aaaccatcta 3960cctcagcatt tactgagcat tttaaaactc agcttcacag agatgtcttt gtgatgtgat 4020gcttagcagt ttggcccgaa gaaggaaaat atccagtacc atgctgtttt gtggcatgaa 4080tatagcccac tgactaggaa ttatttaacc aacccactga aaacttgtgt gtcgagcagc 4140tctgaactga ttttactttt aaagaatttg ctcatggacc tgtcatcctt tttataaaaa 4200ggctcactga caagagacag ctgttaattt cccacagcaa tcattgcaga ctaactttat 4260taggagaagc ctatgccagc tgggagtgat tgctaagagg ctccagtctt tgcattccaa 4320agccttttgc taaagttttg cacttttttt ttttcatttc ccatttttaa gtagttacta 4380agttaactag ttattcttgc ttctgagtat aacgaattgg gatgtctaaa cctattttta 4440tagatgttat ttaaataatg cagcaatatc acctcttatt gacaatacct aaattatgag 4500ttttattaat atttaagact gtaaatggtc ttaaaccact aactactgaa gagctcaatg 4560attgacatct gaaatgcttt gtaattattg acttcagccc ctaagaatgc tatgatttca 4620cgtgcaggtc taatttcaac aggctagagt tagtactact taccagatgt aattatgttt 4680tggaaatgta catattcaaa cagaagtgcc tcattttaga aatgagtagt gctgatggca 4740ctggcacatt acagtggtgt cttgtttaat actcattggt atattccagt agctatctct 4800ctcagttggt ttttgataga acagaggcca gcaaactttc tttgtaaaag gctggttagt 4860aaattattgc aggccacctg tgtctttgtc atacattctt cttgctgttg tttagtttgt 4920tttttttcaa acaaccctct aaaaatgtaa aaaccatgtt tagcttgcag ctgtacaaaa 4980actgcccacc agccagatgt gaccctcagg ccatcatttg ccaatcactg agaattattt 5040ttgttgttgt tgttgttgtt gtttttgaga cagagtctct ctctgttgcc caggctggag 5100tgcagtggcg caatctcagc tcactgcaac ctccgcctcc cgggttcaag cagttctgtc 5160tcagccttct gagtagctgg gactacaggt gcatgccacc acaccctgct aatttttgta 5220tttttagtag agacgggggt tccaccatat tggtcaggct tatcttgaac tcctgacctc 5280aggtgatcca cctgcctctg cctcccaaag tgctgagatt acaggcataa gccagtgcac 5340ccagccgaga attagtattt ttatgtatgg ttaaaccttg gcgtctagcc atattttatg 5400tcataataca atggatttgt gaagagcaga ttccatgagt aactctgaca ggtattttag 5460atcatgatct caacaatatt cctcccaaat ggcatacatc ttttgtacaa agaacttgaa 5520atgtaaatac tgtgtttgtg ctgtaagagt tgtgtatttc aaaaactgaa atctcataaa 5580aagttaaatt ttgaaaa 5597 <210> SEQ ID NO 5 <211> LENGTH: 1219 <212> TYPE:DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:sig_peptide <222> LOCATION: (24)..(95) <223> OTHER INFORMATION: <400>SEQUENCE: 5 gtagagcgca ggtgcgcggc tcgatggcgg cggggctggc gcggctcctgttgctcctcg 60 ggctctcggc cggcgggccc gcgccggcag gtgcagcgaa gatgaaggtggtggaggagc 120 ccaacgcgtt tggggtgaac aacccgttct tgcctcaggc cagtcgcctccaggccaaga 180 gggatccttc acccgtgtct ggacccgtgc atctcttccg actctcgggcaagtgcttca 240 gcctggtgga gtccacgtac aagtatgagt tctgcccgtt ccacaacgtgacccagcacg 300 agcagacctt ccgctggaac gcctacagtg ggatcctcgg catctggcacgagtgggaga 360 tcgccaacaa caccttcacg ggcatgtgga tgagggacgg tgacgcctgccgttcccgga 420 gccggcagag caaggtggag ctggcgtgtg gaaaaagcaa ccggctggcccatgtgtccg 480 agccgagcac ctgcgtctat gcgctgacgt tcgagacccc cctcgtctgccacccccacg 540 ccttgctagt gtacccaacc ctgccagagg ccctgcagcg gcagtgggaccaggtagagc 600 aggacctggc cgatgagctg atcacccccc agggccatga gaagttgctgaggacacttt 660 ttgaggatgc tggctactta aagaccccag aagaaaatga acccacccagctggagggag 720 gtcctgacag cttggggttt gagaccctgg aaaactgcag gaaggctcataaagaactct 780 caaaggagat caaaaggctg aaaggtttgc tcacccagca cggcatcccctacacgaggc 840 ccacagaaac ttccaacttg gagcacttgg gccacgagac gcccagagccaagtctccag 900 agcagctgcg gggtgaccca ggactgcgtg ggagtttgtg accttgtggtgggagagcag 960 aggtggacgc ggccgagagc cctacagaga agctggctgg taggacccgcaggaccagct 1020 gaccaggctt gtgctcagag aagcagacaa aacaaagatt caaggttttaattaattccc 1080 atactgataa aaataactcc atgaattctg taaaccattg cataaatgctatagtgtaaa 1140 aaaatttaaa caagtgttaa ctttaaacag ttcgctacaa gtaaatgattataaatacta 1200 aaaaaaaaaa aaaaaaaaa 1219 <210> SEQ ID NO 6 <211>LENGTH: 515 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: SIGNAL <222> LOCATION: (1)..(24) <223> OTHERINFORMATION: <221> NAME/KEY: PROPEP <222> LOCATION: (25)..(49) <223>OTHER INFORMATION: <400> SEQUENCE: 6 Met Ala Thr Ser Thr Gly Arg Trp LeuLeu Leu Arg Leu Ala Leu Phe 1 5 10 15 Gly Phe Leu Trp Glu Ala Ser GlyGly Leu Asp Ser Gly Ala Ser Arg 20 25 30 Asp Asp Asp Leu Leu Leu Pro TyrPro Arg Ala Arg Ala Arg Leu Pro 35 40 45 Arg Asp Cys Thr Arg Val Arg AlaGly Asn Arg Glu His Glu Ser Trp 50 55 60 Pro Pro Pro Pro Ala Thr Pro GlyAla Gly Gly Leu Ala Val Arg Thr 65 70 75 80 Phe Val Ser His Phe Arg AspArg Ala Val Ala Gly His Leu Thr Arg 85 90 95 Ala Val Glu Pro Leu Arg ThrPhe Ser Val Leu Glu Pro Gly Gly Pro 100 105 110 Gly Gly Cys Ala Ala ArgArg Arg Ala Thr Val Glu Glu Thr Ala Arg 115 120 125 Ala Ala Asp Cys ArgVal Ala Gln Asn Gly Gly Phe Phe Arg Met Asn 130 135 140 Ser Gly Glu CysLeu Gly Asn Val Val Ser Asp Glu Arg Arg Val Ser 145 150 155 160 Ser SerGly Gly Leu Gln Asn Ala Gln Phe Gly Ile Arg Arg Asp Gly 165 170 175 ThrLeu Val Thr Gly Tyr Leu Ser Glu Glu Glu Val Leu Asp Thr Glu 180 185 190Asn Pro Phe Val Gln Leu Leu Ser Gly Val Val Trp Leu Ile Arg Asn 195 200205 Gly Ser Ile Tyr Ile Asn Glu Ser Gln Ala Thr Glu Cys Asp Glu Thr 210215 220 Gln Glu Thr Gly Ser Phe Ser Lys Phe Val Asn Val Ile Ser Ala Arg225 230 235 240 Thr Ala Ile Gly His Asp Arg Lys Gly Gln Leu Val Leu PheHis Ala 245 250 255 Asp Gly His Thr Glu Gln Arg Gly Ile Asn Leu Trp GluMet Ala Glu 260 265 270 Phe Leu Leu Lys Gln Asp Val Val Asn Ala Ile AsnLeu Asp Gly Gly 275 280 285 Gly Ser Ala Thr Phe Val Leu Asn Gly Thr LeuAla Ser Tyr Pro Ser 290 295 300 Asp His Cys Gln Asp Asn Met Trp Arg CysPro Arg Gln Val Ser Thr 305 310 315 320 Val Val Cys Val His Glu Pro ArgCys Gln Pro Pro Asp Cys His Gly 325 330 335 His Gly Thr Cys Val Asp GlyHis Cys Gln Cys Thr Gly His Phe Trp 340 345 350 Arg Gly Pro Gly Cys AspGlu Leu Asp Cys Gly Pro Ser Asn Cys Ser 355 360 365 Gln His Gly Leu CysThr Glu Thr Gly Cys Arg Cys Asp Ala Gly Trp 370 375 380 Thr Gly Ser AsnCys Ser Glu Glu Cys Pro Leu Gly Trp His Gly Pro 385 390 395 400 Gly CysGln Arg Arg Cys Lys Cys Glu His His Cys Pro Cys Asp Pro 405 410 415 LysThr Gly Asn Cys Ser Val Ser Arg Val Lys Gln Cys Leu Gln Pro 420 425 430Pro Glu Ala Thr Leu Arg Ala Gly Glu Leu Ser Phe Phe Thr Arg Thr 435 440445 Ala Trp Leu Ala Leu Thr Leu Ala Leu Ala Phe Leu Leu Leu Ile Ser 450455 460 Ile Ala Ala Asn Leu Ser Leu Leu Leu Ser Arg Ala Glu Arg Asn Arg465 470 475 480 Arg Leu His Gly Asp Tyr Ala Tyr His Pro Leu Gln Glu MetAsn Gly 485 490 495 Glu Pro Leu Ala Ala Glu Lys Glu Gln Pro Gly Gly AlaHis Asn Pro 500 505 510 Phe Lys Asp 515 <210> SEQ ID NO 7 <211> LENGTH:2183 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 7atggcgacct ccacgggtcg ctggcttctc ctccggcttg cactattcgg cttcctctgg 60gaagcgtccg gcggcctcga ctcgggggcc tcccgcgacg acgacttgct actgccctat 120ccacgcgcgc gcgcgcgcct cccccgggac tgcacacggg tgcgcgccgg caaccgcgag 180cacgagagtt ggcctccgcc tcccgcgact cccggcgccg gcggtctggc cgtgcgcacc 240ttcgtgtcgc acttcaggga ccgcgcggtg gccggccacc tgacgcgggc cgttgagccc 300ctgcgcacct tctcggtgct ggagcccggt ggacccggcg gctgcgcggc gagacgacgc 360gccaccgtgg aggagacggc gcgggcggcc gactgccgtg tcgcccagaa cggcggcttc 420ttccgcatga actcgggcga gtgcctgggg aacgtggtga gcgacgagcg gcgggtgagc 480agctccgggg ggctgcagaa cgcgcagttc gggatccgcc gcgacgggac cctggtcacc 540gggtacctgt ctgaggagga ggtgctggac actgagaacc catttgtgca gctgctgagt 600ggggtcgtgt ggctgattcg taatggaagc atctacatca acgagagcca agccacagag 660tgtgacgaga cacaggagac aggttccttt agcaaatttg tgaatgtgat atcagccagg 720acggccattg gccacgaccg gaaagggcag ctggtgctct ttcatgcaga cggccatacg 780gagcagcgtg gcatcaacct gtgggaaatg gcggagttcc tgctgaaaca ggacgtggtc 840aacgccatca acctggatgg gggtggctct gccacctttg tgctcaacgg gaccttggcc 900agttacccgt cagatcactg ccaggacaac atgtggcgct gtccccgcca agtgtccacc 960gtggtgtgtg tgcacgaacc ccgctgccag ccgcctgact gccacggcca cgggacctgc 1020gtggacgggc actgccaatg caccgggcac ttctggcggg gtcccggctg tgatgagctg 1080gactgtggcc cctctaactg cagccagcac ggactgtgca cggagaccgg ctgccgctgt 1140gatgccggat ggaccgggtc caactgcagt gaagagtgtc cccttggctg gcatgggccg 1200ggctgccaga ggcgttgtaa gtgtgagcac cattgtccct gtgaccccaa gactggcaac 1260tgcagcgtct ccagagtaaa gcagtgtctc cagccacctg aagccaccct gagggcggga 1320gaactctcct ttttcaccag gaccgcctgg ctagccctca ccctggcgct ggccttcctc 1380ctgctgatca gcattgcagc aaacctgtcc ttgctcctgt ccagagcaga gaggaaccgg 1440cgcctgcatg gggactatgc ataccacccg ctgcaggaga tgaacgggga gcctctggcc 1500gcagagaagg agcagccagg gggcgcccac aaccccttca aggactgaag cctcaagctg 1560cccggggtgg cacgtcgcga aagcttgttt ccccacggtc tggcttctgc aggggaaatt 1620tcaaggccac tggcgtggac catctgggtg tcctcaatgg cccctgtggg gcagccaagt 1680tcctgatagc acttgtgcct cagcccctca cctggccacc tgccagggca cctgcaaccc 1740tagcaatacc atgctcgctg gagaggctca gctgcctgct tctcgcctgc ctgtgtctgc 1800tgccgagaag cccgtgcccc cgggagggct gccgcactgc caaagagtct ccctcctcct 1860ggggaagggg ctgccaacga accagactca gtgaccacgt catgacagaa cagcacatcc 1920tggccagcac ccctggctgg agtgggttaa agggacgagt ctgccttcct ggctgtgaca 1980cgggacccct tttctacaga cctcatcact ggatttgcca actagaattc gatttcctgt 2040cataggaagc tccttggaag aagggatggg gggatgaaat catgtttaca gacctgtttt 2100gtcatcctgc tgccaagaag ttttttaatc acttgaataa attgatataa taaaaggagc 2160caccaggtgg tgtgtggatt ctg 2183 <210> SEQ ID NO 8 <211> LENGTH: 328 <212>TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 8 Asp Thr Phe AlaAsp Ser Leu Arg Tyr Val Asn Lys Ile Leu Asn Ser 1 5 10 15 Lys Phe GlyPhe Thr Ser Arg Lys Val Pro Ala His Met Pro His Met 20 25 30 Ile Asp ArgIle Val Met Gln Glu Leu Gln Asp Met Phe Pro Glu Glu 35 40 45 Phe Asp LysThr Ser Phe His Lys Val Arg His Ser Glu Asp Met Gln 50 55 60 Phe Ala PheSer Tyr Phe Tyr Tyr Leu Met Ser Ala Val Gln Pro Leu 65 70 75 80 Asn IleSer Gln Val Phe His Glu Val Asp Thr Asp Gln Ser Gly Val 85 90 95 Leu SerAsp Arg Glu Ile Arg Thr Leu Ala Thr Arg Ile His Asp Leu 100 105 110 ProLeu Ser Leu Gln Asp Leu Thr Gly Leu Glu His Met Leu Ile Asn 115 120 125Cys Ser Lys Met Leu Pro Ala Asn Ile Thr Gln Leu Asn Asn Ile Pro 130 135140 Pro Thr Gln Glu Ala Tyr Tyr Asp Pro Asn Leu Pro Pro Val Thr Lys 145150 155 160 Ser Leu Val Thr Asn Cys Lys Pro Val Thr Asp Lys Ile His LysAla 165 170 175 Tyr Lys Asp Lys Asn Lys Tyr Arg Phe Glu Ile Met Gly GluGlu Glu 180 185 190 Ile Ala Phe Lys Met Ile Arg Thr Asn Val Ser His ValVal Gly Gln 195 200 205 Leu Asp Asp Ile Arg Lys Asn Pro Arg Lys Phe ValCys Leu Asn Asp 210 215 220 Asn Ile Asp His Asn His Lys Asp Ala Arg ThrVal Lys Ala Val Leu 225 230 235 240 Arg Asp Phe Tyr Glu Ser Met Phe ProIle Pro Ser Gln Phe Glu Leu 245 250 255 Pro Arg Glu Tyr Arg Asn Arg PheLeu His Met His Glu Leu Gln Glu 260 265 270 Trp Arg Ala Tyr Arg Asp LysLeu Lys Phe Trp Thr His Cys Val Leu 275 280 285 Ala Thr Leu Ile Ile PheThr Ile Phe Ser Phe Phe Ala Glu Gln Ile 290 295 300 Ile Ala Leu Lys ArgLys Ile Phe Pro Arg Arg Arg Ile His Lys Glu 305 310 315 320 Ala Ser ProAsp Arg Ile Arg Val 325 <210> SEQ ID NO 9 <211> LENGTH: 307 <212> TYPE:PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 9 Met Ala Gly Arg LeuAla Gly Phe Leu Met Leu Leu Gly Leu Ala Ser 1 5 10 15 Gln Gly Pro AlaPro Ala Cys Ala Gly Lys Met Lys Val Val Glu Glu 20 25 30 Pro Asn Thr PheGly Leu Asn Asn Pro Phe Leu Pro Gln Ala Ser Arg 35 40 45 Leu Gln Pro LysArg Glu Pro Ser Ala Val Ser Gly Pro Leu His Leu 50 55 60 Phe Arg Leu AlaGly Lys Cys Phe Ser Leu Val Glu Ser Thr Tyr Lys 65 70 75 80 Tyr Glu PheCys Pro Phe His Asn Val Thr Gln His Glu Gln Thr Phe 85 90 95 Arg Trp AsnAla Tyr Ser Gly Ile Leu Gly Ile Trp His Glu Trp Glu 100 105 110 Ile IleAsn Asn Thr Phe Lys Gly Met Trp Met Thr Asp Gly Asp Ser 115 120 125 CysHis Ser Arg Ser Arg Gln Ser Lys Val Glu Leu Thr Cys Gly Lys 130 135 140Ile Asn Arg Leu Ala His Val Ser Glu Pro Ser Thr Cys Val Tyr Ala 145 150155 160 Leu Thr Phe Glu Thr Pro Leu Val Cys His Pro His Ser Leu Leu Val165 170 175 Tyr Pro Thr Leu Ser Glu Ala Leu Gln Gln Arg Leu Asp Gln ValGlu 180 185 190 Gln Asp Leu Ala Asp Glu Leu Ile Thr Pro Gln Gly Tyr GluLys Leu 195 200 205 Leu Arg Val Leu Phe Glu Asp Ala Gly Tyr Leu Lys ValPro Gly Glu 210 215 220 Thr His Pro Thr Gln Leu Ala Gly Gly Ser Lys GlyLeu Gly Leu Glu 225 230 235 240 Thr Leu Asp Asn Cys Arg Lys Ala His AlaGlu Leu Ser Gln Glu Val 245 250 255 Gln Arg Leu Thr Ser Leu Leu Gln GlnHis Gly Ile Pro His Thr Gln 260 265 270 Pro Thr Glu Thr Thr His Ser GlnHis Leu Gly Gln Gln Leu Pro Ile 275 280 285 Gly Ala Ile Ala Ala Glu HisLeu Arg Ser Asp Pro Gly Leu Arg Gly 290 295 300 Asn Ile Leu 305 <210>SEQ ID NO 10 <211> LENGTH: 2070 <212> TYPE: DNA <213> ORGANISM: Musmusculus <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:(186)..(186) <223> OTHER INFORMATION: n is a, t, g, or c <400> SEQUENCE:10 gtgagaccct aggagcaatg gccgggcggc tggctggctt cctgatgttg ctggggctcg 60cgtcgcaggg gcccgcgccg gcatgtgccg ggaagatgaa ggtggtggag gagcctaaca 120cattcgggtg agcggatcac ggtcctgcgg cttggggacc gagcctggct ggttcttctg 180accttntcaa ttccataggc tgaataaccc gttcttgccc caggcaagcc gccttcagcc 240caagagagag ccttcagctg tatcccgcaa attaagagaa attaatttca aacgatttag 300aaagtattct agccaggcga tgatggcgca cgcctttaat cccagcactt gggaggcaga 360ggcaggcaga tttccgagtt caaggccatc agaactgact gtacatctta gtacagttta 420gcatgtgatc agagatctga atcacaaagc tgggcctgcg tggtaaagca ggtcctttct 480aataaggttg cagtttagat tttctttctt aactctttta ttctttgaga cagggtttct 540caacagtggg tgtcctggaa ctcacttttg taaaccaggc tgcccttaaa ctcacaaagc 600tctgtcagcc tctgcctcct gagtgctggg attaaaggtc cacaccctgt tcattcattt 660ttaatttttg agactgggtc tcattatgtg gccctagaca gatactgaga gcctcctcca 720caggaacaag catgggaatc ctgccacaga caaccagttc tgtggtctgg agatgagttt 780gtcagtccct aggagttagg tcagcctgcc tctgcattcc caataattta ggaaaggagc 840ttggggcgtt ctggccttga tggttagtgc cctcctgcca accttagctt ccagctttag 900gggtagcaga gtttataccg atgctaaact gctgttgtgt tcttccccag ggcccctgca 960tctcttcaga cttgctggca agtgctttag cctagtggag tccacgtgag tgccaggctg 1020gtgggtggag tgggcggagt ctgcagagct cctgatgtgc ctgtgtttcc caggtacaag 1080tatgaattct gccctttcca caacgtcacc cagcacgagc agaccttccg ctggaatgcc 1140tacagcggga tccttggcat ctggcatgag tgggaaatca tcaacaatac cttcaagggc 1200atgtggatga ctgatgggga ctcctgccac tcccggagcc ggcagagcaa ggtggagctc 1260acctgtggaa agatcaaccg actggcccac gtgtctgagc caagcacctg tgtctatgca 1320ttgacattcg agacccctct tgtttgccat ccccactctt tgttagtgta tccaactctg 1380tcagaagccc tgcagcagcc cttggaccag gtggaacagg acctggcaga tgaactgatc 1440acaccacagg gctatgagaa gttgctaagg gtactttttg aggatgctgg ctacttaaag 1500gtcccaggag aaacccatcc cacccagctg gcaggaggtt ccaagggcct ggggcttgag 1560actctggaca actgtagaaa ggcacatgca gagctgtcac aggaggtaca aagactgacg 1620agtctgctgc aacagcatgg aatcccccac actcagccca caggtcagtc tgcctgccct 1680ggtcagctgc cagccactcc ggggcctgca gcactggggc agatctttat tgctacccat 1740tctggcagaa accactcact ctcagcacct gggtcagcag ctccccatag gtgcaatcgc 1800agcagagcat ctgcggagtg acccaggact acgtgggaac atcctgtgag caaggtggcc 1860acgaagaata gaaatatcct gagctttgag tgtcctttca cagagtgaac aaaactggtg 1920tggtgtagac acggcttctt ttggcatatt ctagatcaga cagtgtcact gacaaacaag 1980agggacctgc tggccagcct ttgttgtgcc caaagatcca gacaaaataa agattcaaag 2040ttttaattaa aaaaaaaaaa aaaggaattc 2070 <210> SEQ ID NO 11 <211> LENGTH:113 <212> TYPE: PRT <213> ORGANISM: Rattus rattus <400> SEQUENCE: 11 PhePro Pro Thr Phe Lys Glu Thr Ser Gln Leu Lys Thr Lys Leu Pro 1 5 10 15Glu Asn Leu Ser Ser Lys Ile Lys Leu Leu Gln Leu Tyr Ser Glu Ala 20 25 30Ser Val Ala Leu Leu Lys Leu Asn Asn Pro Lys Gly Phe Pro Glu Leu 35 40 45Asn Lys Gln Thr Lys Lys Asn Met Ser Ile Ser Gly Lys Glu Leu Ala 50 55 60Ile Ser Pro Ala Tyr Leu Leu Trp Asp Leu Ser Ala Ile Ser Gln Ser 65 70 7580 Lys Gln Asp Glu Asp Val Ser Ala Ser Arg Phe Glu Asp Asn Glu Glu 85 9095 Leu Arg Tyr Ser Leu Arg Ser Ile Glu Arg His Asp Ser Met Ser Pro 100105 110 Leu <210> SEQ ID NO 12 <211> LENGTH: 460 <212> TYPE: DNA <213>ORGANISM: Rattus rattus <400> SEQUENCE: 12 attcccacca acattcaaggagacgagtca gctgaagaca aaactgccag aaaatctttc 60 ttctaaaata aaactgttgcagctgtactc ggaggccagc gtcgctcttc tgaaattgaa 120 taaccccaaa ggtttccccgagctgaacaa gcagaccaag aagaacatga gcatcagtgg 180 gaaggaactg gccatcagccctgcctatct gctgtgggac ctgagcgcca tcagccagtc 240 caagcaggat gaagatgtgtctgccagccg cttcgaggat aacgaagagc tgaggtactc 300 actgagatct atcgagagacatgattccat gagtccttta tgaattctgg ccatatcttc 360 aatcatgatc tcagtagtattcctctgaaa tggcacacat ttttctaatg agaacttgaa 420 atgtaaatat tgtgtttgtgctgtaaattt tgtgtatttc 460 <210> SEQ ID NO 13 <211> LENGTH: 502 <212>TYPE: PRT <213> ORGANISM: Drosophila melanogaster <400> SEQUENCE: 13 GlyThr Arg Arg Phe Asp Asp Lys Asn Glu Leu Arg Tyr Ser Leu Arg 1 5 10 15Ser Leu Glu Lys His Ala Ala Trp Ile Arg His Val Tyr Ile Val Thr 20 25 30Asn Gly Gln Ile Pro Ser Trp Leu Asp Leu Ser Tyr Glu Arg Val Thr 35 40 45Val Val Pro His Glu Val Leu Ala Pro Asp Pro Asp Gln Leu Pro Thr 50 55 60Phe Ser Ser Ser Ala Ile Glu Thr Phe Leu His Arg Ile Pro Lys Leu 65 70 7580 Ser Lys Arg Phe Leu Tyr Leu Asn Asp Asp Ile Phe Leu Gly Ala Pro 85 9095 Leu Tyr Pro Glu Asp Leu Tyr Thr Glu Ala Glu Gly Val Arg Val Tyr 100105 110 Gln Ala Trp Met Val Pro Gly Cys Ala Leu Asp Cys Pro Trp Thr Tyr115 120 125 Ile Gly Asp Gly Ala Cys Asp Arg His Cys Asn Ile Asp Ala CysGln 130 135 140 Phe Asp Gly Gly Asp Cys Ser Glu Thr Gly Pro Ala Ser AspAla His 145 150 155 160 Val Ile Pro Pro Ser Lys Glu Val Leu Glu Val GlnPro Ala Ala Val 165 170 175 Pro Gln Ser Arg Val His Arg Phe Pro Gln MetGly Leu Gln Lys Leu 180 185 190 Phe Arg Arg Ser Ser Ala Asn Phe Lys AspVal Met Arg His Arg Asn 195 200 205 Val Ser Thr Leu Lys Glu Leu Arg ArgIle Val Glu Arg Phe Asn Lys 210 215 220 Ala Lys Leu Met Ser Leu Asn ProGlu Leu Glu Thr Ser Ser Ser Glu 225 230 235 240 Pro Gln Thr Thr Gln ArgHis Gly Leu Arg Lys Glu Asp Phe Lys Ser 245 250 255 Ser Thr Asp Ile TyrSer His Ser Leu Ile Ala Thr Asn Met Leu Leu 260 265 270 Asn Arg Ala TyrGly Phe Lys Ala Arg His Val Leu Ala His Val Gly 275 280 285 Phe Leu IleAsp Lys Asp Ile Val Glu Ala Met Gln Arg Arg Phe His 290 295 300 Gln GlnIle Leu Asp Thr Ala His Gln Arg Phe Arg Ala Pro Thr Asp 305 310 315 320Leu Gln Tyr Ala Phe Ala Tyr Tyr Ser Phe Leu Met Ser Glu Thr Lys 325 330335 Val Met Ser Val Glu Glu Ile Phe Asp Glu Phe Asp Thr Asp Gly Ser 340345 350 Ala Thr Trp Ser Asp Arg Glu Val Arg Thr Phe Leu Thr Arg Ile Tyr355 360 365 Gln Pro Pro Leu Asp Trp Ser Ala Met Arg Tyr Phe Glu Glu ValVal 370 375 380 Gln Asn Cys Thr Arg Asn Leu Gly Met His Leu Lys Val AspThr Val 385 390 395 400 Glu His Ser Thr Leu Val Tyr Glu Arg Tyr Glu AspSer Asn Leu Pro 405 410 415 Thr Ile Thr Arg Asp Leu Val Val Arg Cys ProLeu Leu Ala Glu Ala 420 425 430 Leu Ala Ala Asn Phe Ala Val Arg Pro LysTyr Asn Phe His Val Ser 435 440 445 Pro Lys Arg Thr Ser His Ser Asn PheMet Met Leu Thr Ser Asn Leu 450 455 460 Thr Glu Val Val Glu Ser Leu AspArg Leu Arg Arg Asn Pro Arg Lys 465 470 475 480 Phe Asn Cys Ile Asn AspAsn Leu Asp Ala Asn Arg Gly Glu Asp Asn 485 490 495 Glu Asp Gly Ala ProSer 500 <210> SEQ ID NO 14 <211> LENGTH: 9792 <212> TYPE: DNA <213>ORGANISM: Mus musculus <400> SEQUENCE: 14 caggctcggg acttactataacacaggaca cttgtcacct gaaagcttga gtcagtcagt 60 tattatggtc tgtgtgtgagatacaagtgg gtgcataggc agtggtgcac acatgtagat 120 cagactttct acagccaattctcttcttcc tcctctccat gggttcaggg tcttcatctc 180 aggttgcaca gcgagttcatttatgtgctg tgccatctcg ccagtcgttc ctatatccta 240 gaggaaaact agtttcttctggtcaagagg aggaaagagt ggagacctgt cattctaaga 300 tacccaaaac agggccaggttggggacctg tgcctttaat cccatcactt ggggattagg 360 tagaagcaag aggctctagaccagtctaca cactgaattt caagccagcc tacctataaa 420 tcagagaccc tgcttcaaaaataaaattaa acaaaaacga agataaacca agctacccaa 480 aacacaagag ttaatccagtcagacaggtc tagcaaatgc taggatgaaa ggtgtgcacc 540 accacgagtg ggctgcaagcctctctctct ctctctctct ctctctctct ctcgtttgtt 600 ttgtttttcg agacaaggtttctctgtgta gccctggctg tcctggaact cactctgtag 660 accaggctgg cctcgagcttcactcttaaa agttcctctt cctcctcctc catcttttcc 720 tcctcttacc ccctaggctccttttcctct tcttgtcttt cagataaagt ctcaagtagt 780 ccagactggt ctcaaactaactaactagcc aagaatagcc aacctcttaa cttccgattc 840 tcctgcctct gctgaatgctggggttgtgg cgtgggccac cacttctggt ttgtgcaaca 900 cagaaggaac tagggctttaagcacgagaa gcaagttctg tacagactta cacaggccca 960 gcatctgttc ttgcaattttctgtaagttt gacataatat gagaataaaa agctatctat 1020 ctcccttcca gccttaccctctctgatgga attcgaatgc gtaatcaaag cacccaacag 1080 cctggcctga aatcacgtggggcaagccca cgtgaccgga gcaccaatcc aatatggcgg 1140 cgcccagggg gcccgggctgttcctcatac ccgcgctgct cggcttactc ggggtggcgt 1200 ggtgcagctt aagcttcgggtgagtgcaag ccgccggggc cagcctggct ggggtccacc 1260 tttcctgagc gctctcaggcacagccctcc gacctcacga tcgccccgtc cctgcagggt 1320 ttcccgcgac gatgacctgctgctgcctta cccactagcg cgcagacgtc cctcgcgaga 1380 ctgcgcccgg gtgcgctcaggtagcccaga gcaggagagc tggcctccgc cacctctggc 1440 cacccacgaa ccccgggcgccaagccacca cgcggccgtg cgcaccttcg tgtcgcactt 1500 cgaggggcgc gcggtggccggccacctgac gcgggtcgcc gatcccctac gcactttctc 1560 ggtgctggag cccggaggagccgggggctg cggcggcaga agcgccgcgg ctactgtgga 1620 ggacacagcc gtccgggccggttgccgcat cgctcagaac ggtggcttct tccgcatgag 1680 cactggcgag tgcttggggaacgtggtgag cgacgggcgg ctggtgagca gctcaggggg 1740 actgcagaac gcgcagttcggtatccgacg cgatggaacc atagtcaccg ggtgaggagg 1800 cagggagccc cggggctgtagagggcaaag ggtctctgat gttctttcag agccatgcct 1860 ccgagtccag gtccctaaccaaacttcctg tctttcttct tccgagtaat gacgctgaca 1920 ccttccttcc tttaagtttattcatgtgcc actgaataat ctgtgatcag gccgtgtgtg 1980 gggacttggg gaggcgaccgtgagcctgaa cacagtttgt gccctagtga actttgtgta 2040 gtattagaga aacatttcgtgttcaacgaa gccatggaac caattggaaa tagtgtagag 2100 tttatggagc agtcccagacagctagctgg aggccttttg ctgtcctgat aaaaatccag 2160 gttagacaag gagcttgttgagggcagcct ttggaagttt ctgtgtttct tgaaatttga 2220 cagcagccag agttgacagcaggcaggcag gagtagaagg tagcgccatc tggtgttcca 2280 gttctcttcc aaggttccgttttttgccaa ggctgggaag tgggctttcc ccaactcttc 2340 tcagcccttg gttgcaatttctgggcctgc ccatgtatct ggttcttcat ccttcaacat 2400 cagccagtgt caccactgttgatcttaggt tttcacagat cctaaaactt ctgccagtga 2460 ccagcgcctg cagtttctcttccctggctc tgtccttcaa cctctctaca ttccagccat 2520 ctccctagct cctctcttggactccctttc agacttgttg tcatgatcac tgtctcagaa 2580 cccctattgc tcctttacaatggtccactg acctgctcac ctcctacttt ttttttttaa 2640 atgtgtgtgc atctgtgtgtgcctgagggg agaccagagt ttgatttcaa atgtcttcta 2700 ttctcttttc ctccatcttattttctaaca caaaatctga atctagagat cactggttca 2760 gttaacctgg ctggccggtaaaccccaggg ccctcctgct tccctctgtc caccccaccc 2820 cagcactaag gctacagtgtgtgctgttcc agccagcttt ctcatgggtg ctgaggatct 2880 gaacgcaggt tcacatgtgtggtgggaagg cttttaccca atgctctgtc tttccagccc 2940 atcctccctt gttaactgccaaacagctgc ctatcctgtc catgtgtagc tcactgctac 3000 ttcttttatt atgaggtcagcacatgttac taaagatggc aagagaagaa ggttctttca 3060 ttgtgtcata gctatagctcaggaggaatt ttatttcctg tgtaggcaca caggagagca 3120 tcttccagct cacactccaactgaactaac tgaacacctg cctatatatc caaagaaggg 3180 gtgtcagtgc caatcacagcacacctccag tgcaaatgaa ggtttgtgtt tgcaccaatc 3240 acagccttgc ctcttttagcatgcatcaca acaaagtcct cctagactat caggggatat 3300 gctctcttgg ccaaggtaggaatagttgca gtgtcatctg gcacaaacca tttcaaacgg 3360 cctggctgag gttatgccttcgggaacctg aagtctttgt gtggttgtct ccaagtgtct 3420 gtggagctcc aggcggctggtgctgacaga cgctttgtct agttggctgt ttgacttttg 3480 cttaagcagc cagggcagtagagtctaaca gatgctaatt tcaggatcag gaagactgta 3540 gaaaaatgag catcaagaagcccctggtac ccaaagctgc tcttgccaat gagtgaacct 3600 ctgccttccc gcttccaggtcctgtcttga agaagaggtt ctggatcccg tgaatccgtt 3660 cgtgcagctg ctgagcggagtcgtgtggct catccgcaat ggaaacatct acatcaacga 3720 gagccaagcc atcgagtgtgacgagacaca ggagacaggt caggaagcac aggtgttctg 3780 ttttatttgt attaggttttgatttgttta ttttgtgcat gcagcgggtg catgcatgct 3840 cctttccttt cgccatgtgagtcctgagta ttgaactcag actgttaagt gtgatgggag 3900 gcactttacc cactgagccactttcccagc cctcagcatc agctttcttc agacccagga 3960 acagtgtgag tgggttattctttagtgttc ccaaacattt actgagcagc tatttactgt 4020 ttagcactat ggtgagagtcctagggattc agtcttatgt agaatataga aggagaatcc 4080 ttggcaataa gctggaaaattgtgacaagt gccaagaaag aaacaggaga aaggggaccg 4140 gtggggacca gaagcacaggtatgaggaaa gtgcctgcag atttgctgta tggtggcctc 4200 cacatggcct aggagtttgtcataaatgca gagccatgag tccaccctcc ctatacctcc 4260 catccagaaa ccactggttaaatcctaaca acttgggtgt gcaggcactc ccttggtgac 4320 tctgatggac actcaaggtcaagggccact tggggatggg ctgatgagtt ggcttggtca 4380 gtaaagtatt tgccttgaaagtgtgaggac ctgagttgga gccccagaaa gaaacattaa 4440 aagccaagtg ctgggatgcacacttgcatt cccagggatg gagctggaag gcagggatag 4500 gcagatccac ggccacacggtgatattcta agctaacaag agacctgtct cacacagaaa 4560 gtgggtggca cctgaggaccaacacccagg gttatcctct gacgtacctc cagagtggaa 4620 aatactgggg tggtggaaaaggacactttg gtcctgggaa tctggctatt cagggtatag 4680 tgtagaggga gagggagactcaagaggctg tctttgagtc aaaggaacaa gctatcagaa 4740 gaactcaggg cagaggcctgtggttcccag gctcagggca gccttcaagg ccctaggcag 4800 agagtagctg ctgggtgaacaagtacagaa gtgaggcctg gggcctcagg caaggcctgt 4860 gaaatccttc caccaacatagaagtttctg gagactgaga tcacatgaag tgcttctggc 4920 tgtggcatgg aagctcactggaggtggagc tgggatgtgg ctcagtgatc cagtgcttgc 4980 cacacgtgca cgagggaaggagccatcaaa agagagaaag tcgggagacc tgaggggtcc 5040 cctggagagc tgggtaaccaccccgggccc ttctccttta ggttctttta gcaaatttgt 5100 gaatgtgatg tcagccaggacagccgtggg tcatgaccgt gaggggcagc ttatcctctt 5160 ccatgctgat ggacagacggaacagcgtgg tgagtcccag gaaccttggg gctgtttgca 5220 cttcagccac cctacctttccagtcggttc tggggtattg gtgggacaag acagctttcc 5280 ggccattttg gaagtttcatctggaggcaa tagcatttac ctactagtga aagaagccag 5340 ttaagccaga gaccacaggggctcaagctg cataccccct ctgcacagcc ttaacctatg 5400 ggagatggca gagttcctgcgtcaacaaga tgtcgtcaat gccatcaacc tggatggagg 5460 cggttctgct acttttgtgctcaatgggac cctggccagt tacccttcag atcactggta 5520 agaacccttg agccacctttgtggctctct cagactgtct cactcagtca atactgagac 5580 cctgttgtgt gccaggccctgggtatccaa aagtgagcag aagagccgag atctcttccc 5640 tcagggtgct gcacagcccatccctggaaa cctgagacag gtcaggaaag gcctccctga 5700 ggacagtgaa gtaagacctgaggagatggc tggccggggt tgagagagcc tttaccggaa 5760 gacaaactgt acgcaatggggaaatccgct aagtggccca gggagaggct ggagctatag 5820 ctcaggagga aaagtacttgcctcgcaagc gaaggacctg agtttaaact ccaaaaccca 5880 tataaaaagc cagatacgagcaagtggcac atgcttgcag tcccagcctt gttgaggaag 5940 agtcaggtga atcctgaccctctggccagc cagcctagcc tactttttgg caaggtccag 6000 gccagcgaga aagataaataaaataaagtt ttaaatgaca tgtatctaag gttgtcctga 6060 ctccatatgc gcacgcacgcatgcacgcac gcacaactgg cagaatggaa agggaggcaa 6120 actggacagc ctttataggctgcggcaggg accagcacca aggcctagac ctcgtctcac 6180 agtgaatccc ccacagccaggacaacatgt ggcgctgtcc ccgccaagtg tccactgtgg 6240 tgtgtgtgca tgaaccgcgctgccagccac ccgactgcag tggccatggg acctgtgtgg 6300 atggccactg tgaatgcaccagccacttct ggcggggcga ggcctgcagc gagctggact 6360 gtggcccctc caactgcagccagcatgggc tgtgcacaga gagtgagtgg ggagcccaca 6420 ggagggtggt gctctggcgggaccccagct cgcccatgct agactcccgc ctgtgtcctt 6480 acccagcctc tgtggtcttgctttggtagc tggctgccac tgtgatgctg ggtggacagg 6540 atccaactgc agtgaaggtgagagctgcct gcaaacactc ctggagaggg tggcctggct 6600 gcacgcagct ggtatgacgccttcgtccct ccttctggct tggaacttac cttcagagcc 6660 ttttctcatt tcgcatgtggatacccgatg ttctacctac tgaaagagcc cacaagtagg 6720 aagccagatt ttcagtattgtcactcaact ctaaggacca atagcaaaaa aacaaagtgg 6780 ccacgcccct gagggagatccaccaaagtc cttaactcct ggaaagcagc tcctggtgat 6840 cctaggcatg ggtagggtggtttcagcatc agctcagtgg agttcccatt cataatttct 6900 tcatcctttt aaggtcataagttctagagc ccaccttaaa tctaggcagt attcttggtg 6960 tttatctgag acaaagtcttatacagccca cgcagttctc taacttagta tgtaaccgag 7020 aatggcctca agcaacctgcttcctccttt caagcgctgg gattataggc atagcaccaa 7080 cttatagggt gctagaagtcaaacccaggg ccctatgtat atgcagcaag cactctagaa 7140 actggaacac agccctgtttgcagcccggt taccttggag ggttgggtcc cagggatctg 7200 agggcatctc cttcagcatggccatgtgca cacccaggag ccaggctgtc tgtgacagga 7260 gaccatgcca cccaaggtgagacctccctg ccaccatctc ctctccacag agtgtcctct 7320 gggctggtat gggccaggttgccagaggcc ctgccagtgt gagcaccagt gtttctgtga 7380 cccgcagact ggcaactgcagcatctccca aggtatgcgg ccttaaaggt tcttgagctg 7440 ggagcccttg gggcaggtctggggtaggtg gactctcccc agcccttctt tctggtgtct 7500 tgcagtgagg cagtgtctccagccaactga ggctacgccg agggcaggag agctggcctc 7560 tttcaccagg taagtgttttagcaggcact gagcccctat gtctcatccg tgaggcacta 7620 gccaggccag gaggtcacaggttaccctct actttgcaag ctcagggaca gtcacaggta 7680 aaactggcat ccaggaaagaccctgagcta cccagtggaa ctcaaaggta gcaggctatg 7740 ggtgtcatgc ctctggctgcagagactcca cttagatgct ggagcagggc catagagaca 7800 ggaaggactc accttatttctgaactcttc cgtgtgttca ggctttgtgt tgttgttgct 7860 tcctttctgc tgtttcctgggtttccagct ccatccccac agggctcatg gaaagaattg 7920 tgaagcaggg ggtgtggctcaattggcaga ttgattgcct ggcatgcaga aagccctagg 7980 ttcaatcccc agcatttcatatcataaccc aggcatggtg gcatcatgtg cctgtaagtc 8040 cagcacttgg gaggtagaagcagaaaagcc acgagtttaa gaatgttagg gagtcttagg 8100 ccaacctggg atacctaagacaagagatag atgtagggag atagattgac agacagacag 8160 acagacagac agacagacagatcttgagct ggaccttctg gcacaagcct gtcatcctag 8220 ctattccagg aagctgaagcaggaagatag caaattcaag gccagcttaa gccacagatt 8280 gagttcaaga tcaacctgagcaactttatg aaatcctatt ataacataaa aagtaggggt 8340 gggaggttag gctgtagctcagtggtagag tgattgccta gcacgcacaa gacccaggtt 8400 caattcccag tactgcaaaaaatatattag gaacccccta aaagcagtaa cattcacatt 8460 agatgtgtgt gtgtgtgtgtgtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgttttg 8520 ttgggtattt atttcatttacatttccaat gctatcccaa aagtccccca catcctcccc 8580 cacccaccac cttgtttttttttttttttt tttttttttt tttgacctga aactcacagg 8640 ttaggttaga caagctgactggtgagctcc aacttccaac gtaccatcat gcctggcttt 8700 tgttttggtg tctctgtgtaaccctggatg tcctggagct ctctctgtag accagcctgg 8760 ccttaaactc acagaaacccacctgtttct gcctcccatg tgctgggatt aaaggcgtgt 8820 gccacctcac ccagccctgctggacttaaa ttgggtcttc attttataag acaagcatga 8880 gctaattccc cagttcctaaaatgttttta acatccttaa acatcagaga ctgtctgtgg 8940 tattccctcc atgtgtcttcagtataccta ctcccctccc tgcctactgg gttcaacatg 9000 cccagtttgg gttctggctgcctgccccca ctcaagactc tcttttccat ctcaggacca 9060 cctggctagc cctcaccctgacactaattt tcctgctgct gatcagcact ggggtcaacg 9120 tgtccttgtt cctgggctccagggccgaga ggaaccggca cctcgacggg gactatgtgt 9180 atcacccact gcaggaggtgaacggggaag cgctgactgc agagaaggag cacatggagg 9240 aaactagcaa ccccttcaaggactgaagag ctgccccaac ggcatgctcc agataatctt 9300 gtccctgctc ctcacttccacaggggacat tgtgaggcca ctggcatgga tgctatgcac 9360 cccacccttt gctggccatattcctcctgt ccccatgctg tggctcatgc caacctagca 9420 ataaggagct ctggagagcctgcacctgcc tcccgctcgc ctatatctgc tgcccagagg 9480 cctgtctcgc acaggggtctcgccactgcc aaagactccc aggaagtcaa agactcccag 9540 taatccacta gcaaatggaactctgtaacg ccatcataac aagagtggcc actctccgcg 9600 tgcacaggta tgaaatataaatccttacac acacacacac acacaccctc ggctcagcca 9660 cggcactcgc cttttatacagcgtcatcgc tggacagcca actagaactc tgcatcctgt 9720 cacaggaagc acctcataagaaggaatggg gagggaaggc agtcgccttg ttttcagacc 9780 ttagccgaat tc 9792<210> SEQ ID NO 15 <211> LENGTH: 908 <212> TYPE: PRT <213> ORGANISM: Musmusculus <400> SEQUENCE: 15 Met Leu Phe Lys Leu Leu Gln Arg Gln Thr TyrThr Cys Leu Ser His 1 5 10 15 Arg Tyr Gly Leu Tyr Val Cys Phe Val GlyVal Val Val Thr Ile Val 20 25 30 Ser Ala Phe Gln Phe Gly Glu Val Val LeuGlu Trp Ser Arg Asp Gln 35 40 45 Tyr His Val Leu Phe Asp Ser Tyr Arg AspAsn Ile Ala Gly Lys Ser 50 55 60 Phe Gln Asn Arg Leu Cys Leu Pro Met ProIle Asp Val Val Tyr Thr 65 70 75 80 Trp Val Asn Gly Thr Asp Leu Glu LeuLeu Lys Glu Leu Gln Gln Val 85 90 95 Arg Glu His Met Glu Glu Glu Gln ArgAla Met Arg Glu Thr Leu Gly 100 105 110 Lys Asn Thr Thr Glu Pro Thr LysLys Ser Glu Lys Gln Leu Glu Cys 115 120 125 Leu Leu Thr His Cys Ile LysVal Pro Met Leu Val Leu Asp Pro Ala 130 135 140 Leu Pro Ala Thr Ile ThrLeu Lys Asp Leu Pro Thr Leu Tyr Pro Ser 145 150 155 160 Phe His Ala SerSer Asp Met Phe Asn Val Ala Lys Pro Lys Asn Pro 165 170 175 Ser Thr AsnVal Pro Val Val Val Phe Asp Thr Thr Lys Asp Val Glu 180 185 190 Asp AlaHis Ala Gly Pro Phe Lys Gly Gly Gln Gln Thr Asp Val Trp 195 200 205 ArgAla Tyr Leu Thr Thr Asp Lys Asp Ala Pro Gly Leu Val Leu Ile 210 215 220Gln Gly Leu Ala Phe Leu Ser Gly Phe Pro Pro Thr Phe Lys Glu Thr 225 230235 240 Ser Gln Leu Lys Thr Lys Leu Pro Arg Lys Ala Phe Pro Leu Lys Ile245 250 255 Lys Leu Leu Arg Leu Tyr Ser Glu Ala Ser Val Ala Leu Leu LysLeu 260 265 270 Asn Asn Pro Lys Gly Phe Gln Glu Leu Asn Lys Gln Thr LysLys Asn 275 280 285 Met Thr Ile Asp Gly Lys Glu Leu Thr Ile Ser Pro AlaTyr Leu Leu 290 295 300 Trp Asp Leu Ser Ala Ile Ser Gln Ser Lys Gln AspGlu Asp Ala Ser 305 310 315 320 Ala Ser Arg Phe Glu Asp Asn Glu Glu LeuArg Tyr Ser Leu Arg Ser 325 330 335 Ile Glu Arg His Ala Pro Trp Val ArgAsn Ile Phe Ile Val Thr Asn 340 345 350 Gly Gln Ile Pro Ser Trp Leu AsnLeu Asp Asn Pro Arg Val Thr Ile 355 360 365 Val Thr His Gln Asp Ile PheGln Asn Leu Ser His Leu Pro Thr Phe 370 375 380 Ser Ser Pro Ala Ile GluSer His Ile His Arg Ile Glu Gly Leu Ser 385 390 395 400 Gln Lys Phe IleTyr Leu Asn Asp Asp Val Met Phe Gly Lys Asp Val 405 410 415 Trp Pro AspAsp Phe Tyr Ser His Ser Lys Gly Gln Lys Val Tyr Leu 420 425 430 Thr TrpPro Val Pro Asn Cys Ala Glu Gly Cys Pro Gly Ser Trp Ile 435 440 445 LysAsp Gly Tyr Cys Asp Lys Ala Cys Asn Thr Ser Pro Cys Asp Trp 450 455 460Asp Gly Gly Asn Cys Ser Gly Asn Thr Ala Gly Asn Arg Phe Val Ala 465 470475 480 Arg Gly Gly Gly Thr Gly Asn Ile Gly Ala Gly Gln His Trp Gln Phe485 490 495 Gly Gly Gly Ile Asn Thr Ile Ser Tyr Cys Asn Gln Gly Cys AlaAsn 500 505 510 Ser Trp Leu Ala Asp Lys Phe Cys Asp Gln Ala Cys Asn ValLeu Ser 515 520 525 Cys Gly Phe Asp Ala Gly Asp Cys Gly Gln Asp His PheHis Glu Leu 530 535 540 Tyr Lys Val Thr Leu Leu Pro Asn Gln Thr His TyrVal Val Pro Lys 545 550 555 560 Gly Glu Tyr Leu Ser Tyr Phe Ser Phe AlaAsn Ile Ala Arg Lys Arg 565 570 575 Ile Glu Gly Thr Tyr Ser Asp Asn ProIle Ile Arg His Ala Ser Ile 580 585 590 Ala Asn Lys Trp Lys Thr Leu HisLeu Ile Met Pro Gly Gly Met Asn 595 600 605 Ala Thr Thr Ile Tyr Phe AsnLeu Thr Leu Gln Asn Ala Asn Asp Glu 610 615 620 Glu Phe Lys Ile Gln IleAla Val Glu Val Asp Thr Arg Glu Ala Pro 625 630 635 640 Lys Leu Asn SerThr Thr Gln Lys Ala Tyr Glu Ser Leu Val Ser Pro 645 650 655 Val Thr ProLeu Pro Gln Ala Asp Val Pro Phe Glu Asp Val Pro Lys 660 665 670 Glu LysArg Phe Pro Lys Ile Arg Arg His Asp Val Asn Ala Thr Gly 675 680 685 ArgPhe Gln Glu Glu Val Lys Ile Pro Arg Val Asn Ile Ser Leu Leu 690 695 700Pro Lys Glu Ala Gln Val Arg Leu Ser Asn Leu Asp Leu Gln Leu Glu 705 710715 720 Arg Gly Asp Ile Thr Leu Lys Gly Tyr Asn Leu Ser Lys Ser Ala Leu725 730 735 Leu Arg Ser Phe Leu Gly Asn Ser Leu Asp Thr Lys Ile Lys ProGln 740 745 750 Ala Arg Thr Asp Glu Thr Lys Gly Asn Leu Glu Val Pro GlnGlu Asn 755 760 765 Pro Ser His Arg Arg Pro His Gly Phe Ala Gly Glu HisArg Ser Glu 770 775 780 Arg Trp Thr Ala Pro Ala Glu Thr Val Thr Val LysGly Arg Asp His 785 790 795 800 Ala Leu Asn Pro Pro Pro Val Leu Glu ThrAsn Ala Arg Leu Ala Gln 805 810 815 Pro Thr Leu Gly Val Thr Val Ser LysGlu Asn Leu Ser Pro Leu Ile 820 825 830 Val Pro Pro Glu Ser His Leu ProLys Glu Glu Glu Ser Asp Arg Ala 835 840 845 Glu Gly Asn Ala Val Pro ValLys Glu Leu Val Pro Gly Arg Arg Leu 850 855 860 Gln Gln Asn Tyr Pro GlyPhe Leu Pro Trp Glu Lys Lys Lys Tyr Phe 865 870 875 880 Gln Asp Leu LeuAsp Glu Glu Glu Ser Leu Lys Thr Gln Leu Ala Tyr 885 890 895 Phe Thr AspArg Lys His Thr Gly Arg Gln Leu Lys 900 905 <210> SEQ ID NO 16 <211>LENGTH: 5229 <212> TYPE: DNA <213> ORGANISM: Mus musculus <400>SEQUENCE: 16 ggcggtgaag gggtgatgct gttcaagctc ctgcagagac agacctatacctgcctatcc 60 cacaggtatg ggctctacgt ctgcttcgtg ggcgtcgttg tcaccatcgtctcggctttc 120 cagttcggag aggtggttct ggaatggagc cgagatcagt accatgttttgtttgattcc 180 tacagagaca acattgctgg gaaatccttt cagaatcggc tctgtctgcccatgccaatc 240 gacgtggttt acacctgggt gaatggcact gaccttgaac tgctaaaggagctacagcag 300 gtccgagagc acatggagga agagcagaga gccatgcggg aaaccctcgggaagaacaca 360 accgaaccga caaagaagag tgagaagcag ctggaatgtc tgctgacgcactgcattaag 420 gtgcccatgc ttgttctgga cccggccctg ccagccacca tcaccctgaaggatctgcca 480 accctttacc catctttcca cgcgtccagc gacatgttca atgttgcgaaaccaaaaaat 540 ccgtctacaa atgtccccgt tgtcgttttt gacactacta aggatgttgaagacgcccat 600 gctggaccgt ttaagggagg ccagcaaaca gatgtttgga gagcctacttgacaacagac 660 aaagacgccc ctggcttagt gctgatacaa ggcttggcgt tcctgagtggattcccaccg 720 accttcaagg agacgagtca actgaagaca aagctgccaa gaaaagctttccctctaaaa 780 ataaagctgt tgcggctgta ctcggaggcc agtgtcgctc ttctgaaattgaataatccc 840 aagggtttcc aagagctgaa caagcagacc aagaagaaca tgaccatcgatgggaaggaa 900 ctgaccatca gccctgcgta tctgctgtgg gacctgagtg ccatcagccagtccaagcag 960 gatgaggacg cgtctgccag ccgctttgag gataatgaag agctgaggtactcgctgcga 1020 tctatcgaga gacacgcgcc atgggtacgg aatattttca ttgtcaccaacgggcagatt 1080 ccatcctggc tgaaccttga caaccctcga gtgaccatag tgacccaccaggacattttc 1140 caaaatctga gccacttgcc tactttcagt tcccctgcta ttgaaagtcacattcaccgc 1200 atcgaagggc tgtcccagaa gtttatttat ctaaatgacg atgtcatgttcggtaaggac 1260 gtctggccgg acgattttta cagccactcc aaaggtcaaa aggtttatttgacatggcct 1320 gtgccaaact gtgcagaggg ctgcccgggc tcctggataa aggacggctattgtgataag 1380 gcctgtaata cctcaccctg tgactgggat ggcggaaact gctctggtaatactgcaggg 1440 aaccggtttg ttgcaagagg tgggggtacc gggaatattg gagctggacagcactggcag 1500 tttggtggag gaataaacac catctcttac tgtaaccaag gatgtgcaaactcctggctg 1560 gctgacaagt tctgtgacca agcctgtaac gtcttatcct gcgggtttgatgctggtgac 1620 tgtggacaag atcattttca tgaattgtat aaagtaacac ttctcccaaaccagactcac 1680 tatgttgtcc ccaaaggtga atacctgtct tatttcagct ttgcaaacatagccagaaaa 1740 agaattgaag ggacctacag cgacaacccc atcatccgcc acgcgtccattgcaaacaag 1800 tggaaaaccc tacacctgat aatgcccggg gggatgaacg ccaccacgatctattttaac 1860 ctcactcttc aaaacgccaa cgacgaagag ttcaagatcc agatagcagtagaggtggac 1920 acgagggagg cgcccaaact gaattctaca acccagaagg cctatgaaagtttggttagc 1980 ccagtgacac ctcttcctca ggctgacgtc ccttttgaag atgtccccaaagagaaacgc 2040 ttccccaaga tcaggagaca tgatgtaaat gcaacaggga gattccaagaggaggtgaaa 2100 atcccccggg taaatatttc actccttccc aaagaggccc aggtgaggctgagcaacttg 2160 gatttgcaac tagaacgtgg agacatcact ctgaaaggat ataacttgtccaagtcagcc 2220 ctgctaaggt ctttcctggg gaattcacta gatactaaaa taaaacctcaagctaggacc 2280 gatgaaacaa aaggcaacct ggaggtccca caggaaaacc cttctcacagacgtccacat 2340 ggctttgctg gtgaacacag atcagagaga tggactgccc cagcagagacagtgaccgtg 2400 aaaggccgtg accacgcttt gaatccaccc ccggtgttgg agaccaatgcaagattggcc 2460 cagcctacac taggcgtgac tgtgtccaaa gagaaccttt caccgctgatcgttccccca 2520 gaaagccact tgccaaaaga agaggagagt gacagggcag aaggcaatgctgtacctgta 2580 aaggagttag tgcctggcag acggttgcag cagaattatc caggctttttgccctgggag 2640 aaaaaaaagt atttccaaga ccttcttgat gaggaagagt cattgaagacccagttggcg 2700 tactttacag accgcaaaca taccgggagg caactaaaag atacatttgcagactccctc 2760 cgatacgtca ataaaattct caacagcaag tttggattca catccaggaaagtccctgca 2820 cacatgccgc acatgattga caggatcgtt atgcaagaac tccaagatatgttccctgaa 2880 gaatttgaca agacttcatt tcacaaggtg cgtcactctg aggacatgcagtttgccttc 2940 tcctactttt attacctcat gagtgcagtt cagcccctca atatttcccaagtctttcat 3000 gaagtagaca cagaccaatc tggtgtcttg tctgataggg aaatccgaacwctggccacg 3060 agaattcacg acctaccttt aagcttgcag gatttgacag gtttggaacacatgttaata 3120 aattgctcaa aaatgctccc cgctaatatc actcaactca acaacatcccaccgactcag 3180 gaagcatact acgaccccaa cctgcctccg gtcactaaga gtcttgtcaccaactgtaag 3240 ccagtaactg acaagatcca caaagcctat aaagacaaga acaaatacaggtttgaaatc 3300 atgggagagg aagaaatcgc tttcaagatg atacgaacca atgtttctcatgtggttggt 3360 cagttggatg acatcagaaa aaaccccagg aagttcgttt gtctgaatgacaacattgac 3420 cacaaccata aagatgcccg gacagtgaag gctgtcctca gggacttctatgagtccatg 3480 tttcccatac cttcccagtt tgagctgcca agagagtatc ggaaccgctttctgcacatg 3540 catgagctcc aagaatggcg ggcatatcga gacaagctga agttttggacccactgcgta 3600 ctagcaacgt tgattatatt tactatattc tcattttttg ctgaacagataattgctctg 3660 aagcgaaaga tatttcccag gaggaggata cacaaagaag ctagtccagaccgaatcagg 3720 gtgtagaaga tcttcatttg aaagtcacct accttagcat ctgtgaacatctccctcctc 3780 gacaccacag cggagtccct gtgatgtggc acagaggcag cctcgtggggagaagggaca 3840 tcgtgcagac cgggttcttc tgcaatggga agagagccca ctgacctggaattattcagc 3900 acactaagaa cctgtgtcaa tagcttgtac agcttgtact tttaaaggatttgccgaagg 3960 acctgtcggc ttgttgacaa accctccctg acaagctgct ggtttcttcccccagttact 4020 gcagactgag aaaccagtcc atcttgaaag caagtgcgga ggggccccagtctttgcatt 4080 ccaaagcttt ccagcataat ttctggcttg tctcctcctt tgatccatttcccatttttt 4140 tttaaaaaac aataagtggc tactaagtta gtcattctca cttctcaaaataacaaatca 4200 ggatgtcaaa acatttgtat agatcttatt taaataatat agaacgattacttctttagc 4260 ctatctaaat tattgatttt tattaacagt caagtggtct tgaaccgctaacaactactg 4320 aagagctcga gattgacgtt gaaagtgctt tgagcttgtt taactcattccccaagaata 4380 ctgtgacctc gtgtgcgggc ctgattgcga agggctagtg tcacgtagcagtgctgctca 4440 ccggatgtaa ttatgtcgtg gaaatgtaca tacagacaaa agtgcctcacttcagaaatg 4500 agtagtgctg atggcaccag cgagtgatgg tgtccatttg gaaacccatgataccttcca 4560 atgcccaccc tgcttacttt atacagagca ggggttaacc aacttctgtcaaagaacagt 4620 aaagaacttg agatacatcc atctttgtca aatagttttc cttgctaacatttattattg 4680 ttggtgtttt gggaggttta ttttatttta ttgctttgtt atttttcaagacggggattc 4740 tctgtgtagc tctggctgtt tggtaattca ctctaaagac caggctggccttgaacttag 4800 agattcacct gcttctgctt cctgaatggt aggacatgtg cccacattgcctacccaccc 4860 cccttttggg gggggtgagc aactcaataa aaagatgaaa acctgctttagtttgcagct 4920 atacaaaagc agcaggcctc agccagactt gacccccggg gccattgttggcccacggga 4980 gaatcatttt tgacgtgggt aagcaaaccc tgatattggt catgctgtgttatgtcatta 5040 tgtggtggtt ttgaattttg gaagatattt tcagtcatga tttcagtagtattcctccaa 5100 aatggcacac atttttgtaa taagaacttg aaatgtaaat attgtgtttgtgctgtaaat 5160 tttgtgtatt tcaaaaactg aagtttcata aaaaaacaca cttattggaaaaaaaaaaaa 5220 aaaaaaaaa 5229 <210> SEQ ID NO 17 <211> LENGTH: 1105<212> TYPE: DNA <213> ORGANISM: Drosophila melanogaster <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (903)..(903) <223> OTHERINFORMATION: n is a, g, c, or t <221> NAME/KEY: misc_feature <222>LOCATION: (935)..(935) <223> OTHER INFORMATION: n is a, g, c, or t <221>NAME/KEY: misc_feature <222> LOCATION: (1023)..(1023) <223> OTHERINFORMATION: n is a, g, c, or t <221> NAME/KEY: misc_feature <222>LOCATION: (1035)..(1035) <223> OTHER INFORMATION: n is a, g, c, or t<221> NAME/KEY: misc_feature <222> LOCATION: (1071)..(1071) <223> OTHERINFORMATION: n is a, g, c, or t <221> NAME/KEY: misc_feature <222>LOCATION: (1100)..(1100) <223> OTHER INFORMATION: n is a, g, c, or t<400> SEQUENCE: 17 ctgcaggaat tcggcacgag gcggttcgat gacaagaatgagctgcggta ctctctgagg 60 tccctggaaa aacacgccgc atggatcagg catgtgtacatagtaaccaa tggccagatt 120 ccaagttggc tggatctcag ctacgaaagg gtcacggtggtgccccacga agtcctggct 180 cccgatcccg accagctgcc caccttctcc agctcggccatcgagacatt tctgcaccgc 240 ataccaaagc tgtccaagag gttcctctac ctcaacgacgacatattcct gggagctccg 300 ctgtatccgg aggacttgta cactgaagcg gagggagttcgcgtgtacca ggcatggatg 360 gtgcccggct gcgccttgga ttgcccctgg acgtacataggtgatggagc ttgcgatcgg 420 cactgcaaca ttgatgcgtg ccaatttgat ggaggcgactgcagtgaaac tgggccagcg 480 agcgatgccc acgtcattcc accaagcaaa gaagtgctcgaggtgcagcc tgccgctgtt 540 ccacaatcaa gagtccaccg atttcctcag atgggtctccaaaagctgtt caggcgcagc 600 tctgccaatt ttaaggatgt tatgcggcac cgcaatgtgtccacactcaa ggaactacgt 660 cgcattgtgg agcgttttaa caaggccaaa ctcatgtcgctgaaccccga actggagacc 720 tccagctccg agccacagac aactcagcgc cacgggctgcgcaaggagga ttttaagtct 780 tccaccgata tttactctca ctcgctgatt gccaccaatatgttgctgaa tagagcctat 840 ggctttaagg cacgccatgt cctggcgcac gtgggcttcctaattgacaa ggatattgtg 900 gangccatgc aacgacgttt taccagcgaa ttctngacactggccattaa cgctttccga 960 gccccaacag atttgcagta cgcattcgct tactacttctttctaatgag cgaaatccaa 1020 gtnatgagtg tagangaaat cttcgatgaa gtcgacaccggacggtttgg ncacctggtc 1080 ggatccagaa gtgcgaaccn tttta 1105 <210> SEQ IDNO 18 <211> LENGTH: 2005 <212> TYPE: DNA <213> ORGANISM: Mus musculus<400> SEQUENCE: 18 gtttcccgcg acgatgacct gctgctgcct tacccactagcgcgcagacg tccctcgcga 60 gactgcgccc gggtgcgctc aggtagccca gagcaggagagctggcctcc gccacctctg 120 gccacccacg aaccccgggc gccaagccac cacgcggccgtgcgcacctt cgtgtcgcac 180 ttcgaggggc gcgcggtggc cggccacctg acgcgggtcgccgatcccct acgcactttc 240 tcggtgctgg agcccggagg agccgggggc tgcggcggcagaagcgccgc ggctactgtg 300 gaggacacag ccgtccgggc cggttgccgc atcgctcagaacggtggctt cttccgcatg 360 agcactggcg agtgcttggg gaacgtggtg agcgacgggcggctggtgag cagctcaggg 420 ggactgcaga acgcgcagtt cggtatccga cgcgatggaaccatagtcac cgggtcctgt 480 cttgaagaag aggttctgga tcccgtgaat ccgttcgtgcagctgctgag cggagtcgtg 540 tggctcatcc gcaatggaaa catctacatc aacgagagccaagccatcga gtgtgacgag 600 acacaggaga caggttcttt tagcaaattt gtgaatgtgatgtcagccag gacagccgtg 660 ggtcatgacc gtgaggggca gcttatcctc ttccatgctgatggacagac ggaacagcgt 720 ggccttaacc tatgggagat ggcagagttc ctgcgtcaacaagatgtcgt caatgccatc 780 aacctggatg gaggcggttc tgctactttt gtgctcaatgggaccctggc cagttaccct 840 tcagatcact gccaggacaa catgtggcgc tgtccccgccaagtgtccac tgtggtgtgt 900 gtgcatgaac cgcgctgcca gccacccgac tgcagtggccatgggacctg tgtggatggc 960 cactgtgaat gcaccagcca cttctggcgg ggcgaggcctgcagcgagct ggactgtggc 1020 ccctccaact gcagccagca tgggctgtgc acagctggctgccactgtga tgctgggtgg 1080 acaggatcca actgcagtga agagtgtcct ctgggctggtatgggccagg ttgccagagg 1140 ccctgccagt gtgagcacca gtgtttctgt gacccgcagactggcaactg cagcatctcc 1200 caagtgaggc agtgtctcca gccaactgag gctacgccgagggcaggaga gctggcctct 1260 ttcaccagga ccacctggct agccctcacc ctgacactaattttcctgct gctgatcagc 1320 actggggtca acgtgtcctt gttcctgggc tccagggccgagaggaaccg gcacctcgac 1380 ggggactatg tgtatcaccc actgcaggag gtgaacggggaagcgctgac tgcagagaag 1440 gagcacatgg aggaaactag caaccccttc aaggactgaagagctgcccc aacggcatgc 1500 tccagataat cttgtccctg ctcctcactt ccacaggggacattgtgagg ccactggcat 1560 ggatgctatg caccccaccc tttgctggcc atattcctcctgtccccatg ctgtggctca 1620 tgccaaccta gcaataagga gctctggaga gcctgcacctgcctcccgct cgcctatatc 1680 tgctgcccag aggcctgtct cgcacagggg tctcgccactgccaaagact cccaggaagt 1740 caaagactcc cagtaatcca ctagcaaatg gaactctgtaacgccatcat aacaagagtg 1800 gccactctcc gcgtgcacag gtatgaaata taaatccttacacacacaca cacacacacc 1860 ctcggctcag ccacggcact cgccttttat acagcgtcatcgctggacag ccaactagaa 1920 ctctgcatcc tgtcacagga agcacctcat aagaaggaatggggagggaa ggcagtcgcc 1980 ttgttttcag accttagccg aattc 2005 <210> SEQ IDNO 19 <211> LENGTH: 492 <212> TYPE: PRT <213> ORGANISM: Mus musculus<400> SEQUENCE: 19 Val Ser Arg Asp Asp Asp Leu Leu Leu Pro Tyr Pro LeuAla Arg Arg 1 5 10 15 Arg Pro Ser Arg Asp Cys Ala Arg Val Arg Ser GlySer Pro Glu Gln 20 25 30 Glu Ser Trp Pro Pro Pro Pro Leu Ala Thr His GluPro Arg Ala Pro 35 40 45 Ser His His Ala Ala Val Arg Thr Phe Val Ser HisPhe Glu Gly Arg 50 55 60 Ala Val Ala Gly His Leu Thr Arg Val Ala Asp ProLeu Arg Thr Phe 65 70 75 80 Ser Val Leu Glu Pro Gly Gly Ala Gly Gly CysGly Gly Arg Ser Ala 85 90 95 Ala Ala Thr Val Glu Asp Thr Ala Val Arg AlaGly Cys Arg Ile Ala 100 105 110 Gln Asn Gly Gly Phe Phe Arg Met Ser ThrGly Glu Cys Leu Gly Asn 115 120 125 Val Val Ser Asp Gly Arg Leu Val SerSer Ser Gly Gly Leu Gln Asn 130 135 140 Ala Gln Phe Gly Ile Arg Arg AspGly Thr Ile Val Thr Gly Ser Cys 145 150 155 160 Leu Glu Glu Glu Val LeuAsp Pro Val Asn Pro Phe Val Gln Leu Leu 165 170 175 Ser Gly Val Val TrpLeu Ile Arg Asn Gly Asn Ile Tyr Ile Asn Glu 180 185 190 Ser Gln Ala IleGlu Cys Asp Glu Thr Gln Glu Thr Gly Ser Phe Ser 195 200 205 Lys Phe ValAsn Val Met Ser Ala Arg Thr Ala Val Gly His Asp Arg 210 215 220 Glu GlyGln Leu Ile Leu Phe His Ala Asp Gly Gln Thr Glu Gln Arg 225 230 235 240Gly Leu Asn Leu Trp Glu Met Ala Glu Phe Leu Arg Gln Gln Asp Val 245 250255 Val Asn Ala Ile Asn Leu Asp Gly Gly Gly Ser Ala Thr Phe Val Leu 260265 270 Asn Gly Thr Leu Ala Ser Tyr Pro Ser Asp His Cys Gln Asp Asn Met275 280 285 Trp Arg Cys Pro Arg Gln Val Ser Thr Val Val Cys Val His GluPro 290 295 300 Arg Cys Gln Pro Pro Asp Cys Ser Gly His Gly Thr Cys ValAsp Gly 305 310 315 320 His Cys Glu Cys Thr Ser His Phe Trp Arg Gly GluAla Cys Ser Glu 325 330 335 Leu Asp Cys Gly Pro Ser Asn Cys Ser Gln HisGly Leu Cys Thr Ala 340 345 350 Gly Cys His Cys Asp Ala Gly Trp Thr GlySer Asn Cys Ser Glu Glu 355 360 365 Cys Pro Leu Gly Trp Tyr Gly Pro GlyCys Gln Arg Pro Cys Gln Cys 370 375 380 Glu His Gln Cys Phe Cys Asp ProGln Thr Gly Asn Cys Ser Ile Ser 385 390 395 400 Gln Val Arg Gln Cys LeuGln Pro Thr Glu Ala Thr Pro Arg Ala Gly 405 410 415 Glu Leu Ala Ser PheThr Arg Thr Thr Trp Leu Ala Leu Thr Leu Thr 420 425 430 Leu Ile Phe LeuLeu Leu Ile Ser Thr Gly Val Asn Val Ser Leu Phe 435 440 445 Leu Gly SerArg Ala Glu Arg Asn Arg His Leu Asp Gly Asp Tyr Val 450 455 460 Tyr HisPro Leu Gln Glu Val Asn Gly Glu Ala Leu Thr Ala Glu Lys 465 470 475 480Glu His Met Glu Glu Thr Ser Asn Pro Phe Lys Asp 485 490 <210> SEQ ID NO20 <211> LENGTH: 3783 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400>SEQUENCE: 20 gccaccatgg ggttcaagct cttgcagaga caaacctata cctgcctgtcccacaggtat 60 gggctctacg tgtgcttctt gggcgtcgtt gtcaccatcg tctccgccttccagttcgga 120 gaggtggttc tggaatggag ccgagatcaa taccatgttt tgtttgattcctatagagac 180 aatattgctg gaaagtcctt tcagaatcgg ctttgtctgc ccatgccgattgacgttgtt 240 tacacctggg tgaatggcac agatcttgaa ctactgaagg aactacagcaggtcagagaa 300 cagatggagg aggagcagaa agcaatgaga gaaatccttg ggaaaaacacaacggaacct 360 actaagaaga gtgagaagca gttagagtgt ttgctaacac actgcattaaggtgccaatg 420 cttgtcctgg acccagccct gccagccaac atcaccctga aggacctgccatctctttat 480 ccttcttttc attctgccag tgacattttc aatgttgcaa aaccaaaaaacccttctacc 540 aatgtctcag ttgttgtttt tgacagtact aaggatgttg aagatgcccactctggactg 600 cttaaaggaa atagcagaca gacagtatgg aggggctact tgacaacagataaagaagtc 660 cctggattag tgctaatgca agatttggct ttcctgagtg gatttccaccaacattcaag 720 gaaacaaatc aactaaaaac aaaattgcca gaaaatcttt cctctaaagtcaaactgttg 780 cagttgtatt cagaggccag tgtagcgctt ctaaaactga ataaccccaaggattttcaa 840 gaattgaata agcaaactaa gaagaacatg accattgatg gaaaagaactgaccataagt 900 cctgcatatt tattatggga tctgagcgcc atcagccagt ctaagcaggatgaagacatc 960 tctgccagtc gttttgaaga taacgaagaa ctgaggtact cattgcgatctatcgagagg 1020 catgcaccat gggttcggaa tattttcatt gtcaccaacg ggcagattccatcctggctg 1080 aaccttgaca atcctcgagt gacaatagta acacaccagg atgtttttcgaaatttgagc 1140 cacttgccta cctttagttc acctgctatt gaaagtcacg ttcatcgcatcgaagggctg 1200 tcccagaagt ttatttacct aaatgatgat gtcatgtttg ggaaggatgtctggccagat 1260 gatttttaca gtcactccaa aggccagaag gtttatttga catggcctgtgccaaactgt 1320 gccgagggct gcccaggttc ctggattaag gatggctatt gtgacaaggcttgtaataat 1380 tcagcctgcg attgggatgg tggggattgc tctggaaaca gtggagggagtcgctatatt 1440 gcaggaggtg gaggtactgg gagtattgga gttggacagc cctggcagtttggtggagga 1500 ataaacagtg tctcttactg taatcaggga tgtgcgaatt cctggctcgctgataagttc 1560 tgtgaccaag catgcaatgt cttgtcctgt gggtttgatg ctggcgactgtgggcaagat 1620 cattttcatg aattgtataa agtgatcctt ctcccaaacc agactcactatattattcca 1680 aaaggtgaat gcctgcctta tttcagcttt gcagaagtag ccaaaagaggagttgaaggt 1740 gcctatagtg acaatccaat aattcgacat gcttctattg ccaacaagtggaaaaccatc 1800 cacctcataa tgcacagtgg aatgaatgcc accacaatac attttaatctcacgtttcaa 1860 aatacaaacg atgaagagtt caaaatgcag ataacagtgg aggtggacacaagggaggga 1920 ccaaaactga attctacggc ccagaagggt tacgaaaatt tagttagtcccataacactt 1980 cttccagagg cggaaatcct ttttgaggat attcccaaag aaaaacgcttcccgaagttt 2040 aagagacatg atgttaactc aacaaggaga gcccaggaag aggtgaaaattcccctggta 2100 aatatttcac tccttccaaa agacgcccag ttgagtctca ataccttggatttgcaactg 2160 gaacatggag acatcacttt gaaaggatac aatttgtcca agtcagccttgctgagatca 2220 tttctgatga actcacagca tgctaaaata aaaaatcaag ctataataacagatgaaaca 2280 aatgacagtt tggtggctcc acaggaaaaa caggttcata aaagcatcttgccaaacagc 2340 ttaggagtgt ctgaaagatt gcagaggttg acttttcctg cagtgagtgtaaaagtgaat 2400 ggtcatgacc agggtcagaa tccacccctg gacttggaga ccacagcaagatttagagtg 2460 gaaactcaca cccaaaaaac cataggcgga aatgtgacaa aagaaaagcccccatctctg 2520 attgttccac tggaaagcca gatgacaaaa gaaaagaaaa tcacagggaaagaaaaagag 2580 aacagtagaa tggaggaaaa tgctgaaaat cacataggcg ttactgaagtgttacttgga 2640 agaaagctgc agcattacac agatagttac ttgggctttt tgccatgggagaaaaaaaag 2700 tatttcctag atcttctcga cgaagaagag tcattgaaga cacaattggcatacttcact 2760 gatagcaaga atactgggag gcaactaaaa gatacatttg cagattccctcagatatgta 2820 aataaaattc taaatagcaa gtttggattc acatcgcgga aagtccctgctcacatgcct 2880 cacatgattg accggattgt tatgcaagaa ctgcaagata tgttccctgaagaatttgac 2940 aagacgtcat ttcacaaagt gcgccattct gaggatatgc agtttgccttctcttatttt 3000 tattatctca tgagtgcagt gcagccactg aatatatctc aagtctttgatgaagttgat 3060 acagatcaat ctggtgtctt gtctgacaga gaaatccgaa cactggctaccagaattcac 3120 gaactgccgt taagtttgca ggatttgaca ggtctggaac acatgctaataaattgctca 3180 aaaatgcttc ctgctgatat cacgcagcta aataatattc caccaactcaggaatcctac 3240 tatgatccca acctgccacc ggtcactaaa agtctagtaa caaactgtaaaccagtaact 3300 gacaaaatcc acaaagcata taaggacaaa aacaaatata ggtttgaaatcatgggagaa 3360 gaagaaatcg cttttaaaat gattcgtacc aacgtttctc atgtggttggccagttggat 3420 gacataagaa aaaaccctag gaagtttgtt tgcctgaatg acaacattgaccacaatcat 3480 aaagatgctc agacagtgaa ggctgttctc agggacttct atgaatccatgttccccata 3540 ccttcccaat ttgaactgcc aagagagtat cgaaaccgtt tccttcatatgcatgagctg 3600 caggaatgga gggcttatcg agacaaattg aagttttgga cccattgtgtactagcaaca 3660 ttgattatgt ttactatatt ctcatttttt gctgagcagt taattgcacttaagcggaag 3720 atatttccca gaaggaggat acacaaagaa gctagtccca atcgaatcagagtatagaag 3780 atc 3783 <210> SEQ ID NO 21 <211> LENGTH: 3621 <212>TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 21 ctagccgccaccatggagac agacacactc ctgctatggg tactgctgct ctgggttcca 60 ggttccactggtgacgaaga tcaggtagat ccgcggttaa tcgacggtaa gcttagccga 120 gatcaataccatgttttgtt tgattcctat agagacaata ttgctggaaa gtcctttcag 180 aatcggctttgtctgcccat gccgattgac gttgtttaca cctgggtgaa tggcacagat 240 cttgaactactgaaggaact acagcaggtc agagaacaga tggaggagga gcagaaagca 300 atgagagaaatccttgggaa aaacacaacg gaacctacta agaagagtga gaagcagtta 360 gagtgtttgctaacacactg cattaaggtg ccaatgcttg tcctggaccc agccctgcca 420 gccaacatcaccctgaagga cctgccatct ctttatcctt cttttcattc tgccagtgac 480 attttcaatgttgcaaaacc aaaaaaccct tctaccaatg tctcagttgt tgtttttgac 540 agtactaaggatgttgaaga tgcccactct ggactgctta aaggaaatag cagacagaca 600 gtatggaggggctacttgac aacagataaa gaagtccctg gattagtgct aatgcaagat 660 ttggctttcctgagtggatt tccaccaaca ttcaaggaaa caaatcaact aaaaacaaaa 720 ttgccagaaaatctttcctc taaagtcaaa ctgttgcagt tgtattcaga ggccagtgta 780 gcgcttctaaaactgaataa ccccaaggat tttcaagaat tgaataagca aactaagaag 840 aacatgaccattgatggaaa agaactgacc ataagtcctg catatttatt atgggatctg 900 agcgccatcagccagtctaa gcaggatgaa gacatctctg ccagtcgttt tgaagataac 960 gaagaactgaggtactcatt gcgatctatc gagaggcatg caccatgggt tcggaatatt 1020 ttcattgtcaccaacgggca gattccatcc tggctgaacc ttgacaatcc tcgagtgaca 1080 atagtaacacaccaggatgt ttttcgaaat ttgagccact tgcctacctt tagttcacct 1140 gctattgaaagtcacgttca tcgcatcgaa gggctgtccc agaagtttat ttacctaaat 1200 gatgatgtcatgtttgggaa ggatgtctgg ccagatgatt tttacagtca ctccaaaggc 1260 cagaaggtttatttgacatg gcctgtgcca aactgtgccg agggctgccc aggttcctgg 1320 attaaggatggctattgtga caaggcttgt aataattcag cctgcgattg ggatggtggg 1380 gattgctctggaaacagtgg agggagtcgc tatattgcag gaggtggagg tactgggagt 1440 attggagttggacagccctg gcagtttggt ggaggaataa acagtgtctc ttactgtaat 1500 cagggatgtgcgaattcctg gctcgctgat aagttctgtg accaagcatg caatgtcttg 1560 tcctgtgggtttgatgctgg cgactgtggg caagatcatt ttcatgaatt gtataaagtg 1620 atccttctcccaaaccagac tcactatatt attccaaaag gtgaatgcct gccttatttc 1680 agctttgcagaagtagccaa aagaggagtt gaaggtgcct atagtgacaa tccaataatt 1740 cgacatgcttctattgccaa caagtggaaa accatccacc tcataatgca cagtggaatg 1800 aatgccaccacaatacattt taatctcacg tttcaaaata caaacgatga agagttcaaa 1860 atgcagataacagtggaggt ggacacaagg gagggaccaa aactgaattc tacggcccag 1920 aagggttacgaaaatttagt tagtcccata acacttcttc cagaggcgga aatccttttt 1980 gaggatattcccaaagaaaa acgcttcccg aagtttaaga gacatgatgt taactcaaca 2040 aggagagcccaggaagaggt gaaaattccc ctggtaaata tttcactcct tccaaaagac 2100 gcccagttgagtctcaatac cttggatttg caactggaac atggagacat cactttgaaa 2160 ggatacaatttgtccaagtc agccttgctg agatcatttc tgatgaactc acagcatgct 2220 aaaataaaaaatcaagctat aataacagat gaaacaaatg acagtttggt ggctccacag 2280 gaaaaacaggttcataaaag catcttgcca aacagcttag gagtgtctga aagattgcag 2340 aggttgacttttcctgcagt gagtgtaaaa gtgaatggtc atgaccaggg tcagaatcca 2400 cccctggacttggagaccac agcaagattt agagtggaaa ctcacaccca aaaaaccata 2460 ggcggaaatgtgacaaaaga aaagccccca tctctgattg ttccactgga aagccagatg 2520 acaaaagaaaagaaaatcac agggaaagaa aaagagaaca gtagaatgga ggaaaatgct 2580 gaaaatcacataggcgttac tgaagtgtta cttggaagaa agctgcagca ttacacagat 2640 agttacttgggctttttgcc atgggagaaa aaaaagtatt tcctagatct tctcgacgaa 2700 gaagagtcattgaagacaca attggcatac ttcactgata gcaagaatac tgggaggcaa 2760 ctaaaagatacatttgcaga ttccctcaga tatgtaaata aaattctaaa tagcaagttt 2820 ggattcacatcgcggaaagt ccctgctcac atgcctcaca tgattgaccg gattgttatg 2880 caagaactgcaagatatgtt ccctgaagaa tttgacaaga cgtcatttca caaagtgcgc 2940 cattctgaggatatgcagtt tgccttctct tatttttatt atctcatgag tgcagtgcag 3000 ccactgaatatatctcaagt ctttgatgaa gttgatacag atcaatctgg tgtcttgtct 3060 gacagagaaatccgaacact ggctaccaga attcacgaac tgccgttaag tttgcaggat 3120 ttgacaggtctggaacacat gctaataaat tgctcaaaaa tgcttcctgc tgatatcacg 3180 cagctaaataatattccacc aactcaggaa tcctactatg atcccaacct gccaccggtc 3240 actaaaagtctagtaacaaa ctgtaaacca gtaactgaca aaatccacaa agcatataag 3300 gacaaaaacaaatataggtt tgaaatcatg ggagaagaag aaatcgcttt taaaatgatt 3360 cgtaccaacgtttctcatgt ggttggccag ttggatgaca taagaaaaaa ccctaggaag 3420 tttgtttgcctgaatgacaa cattgaccac aatcataaag atgctcagac agtgaaggct 3480 gttctcagggacttctatga atccatgttc cccatacctt cccaatttga actgccaaga 3540 gagtatcgaaaccgtttcct tcatatgcat gagctgcagg aatggagggc ttatcgagac 3600 aaattgaagtagtagtctag a 3621 <210> SEQ ID NO 22 <211> LENGTH: 1383 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <400> SEQUENCE: 22 atggcgacct ccacgggtcgctggcttctc ctccggcttg cactattcgg cttcctctgg 60 gaagcgtccg gcggcctcgactcgggggcc tcccgcgacg acgacttgct actgccctat 120 ccacgcgcgc gcgcgcgcctcccccgggac tgcacacggg tgcgcgccgg caaccgcgag 180 cacgagagtt ggcctccgcctcccgcgact cccggcgccg gcggtctggc cgtgcgcacc 240 ttcgtgtcgc acttcagggaccgcgcggtg gccggccacc tgacgcgggc cgttgagccc 300 ctgcgcacct tctcggtgctggagcccggt ggacccggcg gctgcgcggc gagacgacgc 360 gccaccgtgg aggagacggcgcgggcggcc gactgccgtg tcgcccagaa cggcggcttc 420 ttccgcatga actcgggcgagtgcctgggg aacgtggtga gcgacgagcg gcgggtgagc 480 agctccgggg ggctgcagaacgcgcagttc gggatccgcc gcgacgggac cctggtcacc 540 gggtacctgt ctgaggaggaggtgctggac actgagaacc catttgtgca gctgctgagt 600 ggggtcgtgt ggctgattcgtaatggaagc atctacatca acgagagcca agccacagag 660 tgtgacgaga cacaggagacaggttccttt agcaaatttg tgaatgtgat atcagccagg 720 acggccattg gccacgaccggaaagggcag ctggtgctct ttcatgcaga cggccatacg 780 gagcagcgtg gcatcaacctgtgggaaatg gcggagttcc tgctgaaaca ggacgtggtc 840 aacgccatca acctggatgggggtggctct gccacctttg tgctcaacgg gaccttggcc 900 agttacccgt cagatcactgccaggacaac atgtggcgct gtccccgcca agtgtccacc 960 gtggtgtgtg tgcacgaaccccgctgccag ccgcctgact gccacggcca cgggacctgc 1020 gtggacgggc actgccaatgcaccgggcac ttctggcggg gtcccggctg tgatgagctg 1080 gactgtggcc cctctaactgcagccagcac ggactgtgca cggagaccgg ctgccgctgt 1140 gatgccggat ggaccgggtccaactgcagt gaagagtgtc cccttggctg gcatgggccg 1200 ggctgccaga ggccttgtaagtgtgagcac cattgtccct gtgaccccaa gactggcaac 1260 tgcagcgtct ccagagtaaagcagtgtctc cagccacctg aagccaccct gagggcggga 1320 gaactctcct ttttcaccagggaggaccag gtggacccca ggctgatcga cggcaaggat 1380 tga 1383 <210> SEQ IDNO 23 <211> LENGTH: 32 <212> TYPE: PRT <213> ORGANISM: Homo sapiens<220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (2)..(2)<223> OTHER INFORMATION: Xaa is any amino acid <400> SEQUENCE: 23 AspXaa Thr Arg Val His Ala Gly Arg Leu Glu His Glu Ser Trp Pro 1 5 10 15Pro Ala Ala Gln Thr Ala Gly Ala His Arg Pro Ser Val Arg Thr Phe 20 25 30<210> SEQ ID NO 24 <211> LENGTH: 20 <212> TYPE: PRT <213> ORGANISM: Bostaurus <400> SEQUENCE: 24 Arg Asp Gly Thr Leu Val Thr Gly Tyr Leu SerGlu Glu Glu Val Leu 1 5 10 15 Asp Thr Glu Asn 20 <210> SEQ ID NO 25<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Bos taurus <400>SEQUENCE: 25 Gly Ile Asn Leu Trp Glu Met Ala Glu Phe Leu Leu Lys 1 5 10<210> SEQ ID NO 26 <211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Bostaurus <400> SEQUENCE: 26 Met Leu Leu Lys Leu Leu Gln Arg Gln Arg GlnThr Tyr 1 5 10 <210> SEQ ID NO 27 <211> LENGTH: 28 <212> TYPE: PRT <213>ORGANISM: Bos taurus <400> SEQUENCE: 27 Asp Thr Phe Ala Asp Ser Leu ArgTyr Val Asn Lys Ile Leu Asn Ser 1 5 10 15 Lys Phe Gly Phe Thr Ser ArgLys Val Pro Ala His 20 25 <210> SEQ ID NO 28 <211> LENGTH: 21 <212>TYPE: PRT <213> ORGANISM: Bos taurus <400> SEQUENCE: 28 Ala Lys Met LysVal Val Glu Glu Pro Asn Thr Phe Gly Leu Asn Asn 1 5 10 15 Pro Phe LeuPro Gln 20 <210> SEQ ID NO 29 <211> LENGTH: 5 <212> TYPE: PRT <213>ORGANISM: Bos taurus <400> SEQUENCE: 29 Ile Leu Asn Ser Lys 1 5 <210>SEQ ID NO 30 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Bos taurus<400> SEQUENCE: 30 Thr Ser Phe His Lys 1 5 <210> SEQ ID NO 31 <211>LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Bos taurus <400> SEQUENCE: 31Phe Gly Phe Thr Ser Arg 1 5 <210> SEQ ID NO 32 <211> LENGTH: 12 <212>TYPE: PRT <213> ORGANISM: Bos taurus <400> SEQUENCE: 32 Ser Leu Val ThrAsn Cys Lys Pro Val Thr Asp Lys 1 5 10 <210> SEQ ID NO 33 <211> LENGTH:12 <212> TYPE: PRT <213> ORGANISM: Bos taurus <400> SEQUENCE: 33 Leu AlaHis Val Ser Glu Pro Ser Thr Cys Val Tyr 1 5 10 <210> SEQ ID NO 34 <211>LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Bos taurus <400> SEQUENCE: 34Asn Asn Pro Phe Leu Pro Gln Thr Ser Arg Leu Gln Pro 1 5 10 <210> SEQ IDNO 35 <211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM: Bos taurus <220>FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (8)..(8) OTHERINFORMATION: Xaa is any amino acid NAME/KEY: misc_feature <222>LOCATION: (10)..(10) <223> OTHER INFORMATION: Xaa is any amino acid<221> NAME/KEY: misc_feature <222> LOCATION: (13)..(13) <223> OTHERINFORMATION: Xaa is any amino acid <221> NAME/KEY: misc_feature <222>LOCATION: (15)..(15) <223> OTHER INFORMATION: Xaa is any amino acidSEQUENCE: 35 Val Pro Met Leu Val Leu Asp Xaa Ala Xaa Pro Thr Xaa Val XaaLeu 1 5 10 15 Lys <210> SEQ ID NO 36 <211> LENGTH: 22 <212> TYPE: PRT<213> ORGANISM: Bos taurus <400> SEQUENCE: 36 Glu Leu Pro Ser Leu TyrPro Ser Phe Leu Ser Ala Ser Asp Val Phe 1 5 10 15 Asn Val Ala Lys ProLys 20 <210> SEQ ID NO 37 <211> LENGTH: 25 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:synthetic DNA <400> SEQUENCE: 37 gcgaagatga aggtggtgga ggacc 25 <210>SEQ ID NO 38 <211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: synthetic DNA <400>SEQUENCE: 38 tgcagagaca gacctatacc tgcc 24 <210> SEQ ID NO 39 <211>LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: synthetic DNA <400> SEQUENCE: 39actcacctct ccgaactgga aag 23 <210> SEQ ID NO 40 <211> LENGTH: 29 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: synthetic DNA <400> SEQUENCE: 40 ctagccacca tggggttcaagctcttgca 29 <210> SEQ ID NO 41 <211> LENGTH: 21 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:synthetic DNA <400> SEQUENCE: 41 agagcttgaa ccccatggtg g 21 <210> SEQ IDNO 42 <211> LENGTH: 60 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: synthetic DNA <400>SEQUENCE: 42 gaagacacaa ttggcatact tcactgatag caagaatact gggaggcaactaaaagatac 60 <210> SEQ ID NO 43 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:synthetic DNA <400> SEQUENCE: 43 actgcatatc ctcagaatgg 20 <210> SEQ IDNO 44 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: synthetic DNA <400>SEQUENCE: 44 tggttctgaa gcttagccga gatcaatacc atg 33 <210> SEQ ID NO 45<211> LENGTH: 40 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: synthetic DNA <400> SEQUENCE: 45tagtacactc tagactacta cttcaatttg tctcgataag 40 <210> SEQ ID NO 46 <211>LENGTH: 218 <212> TYPE: DNA <213> ORGANISM: hybrid <220> FEATURE: <221>NAME/KEY: misc_feature <223> OTHER INFORMATION: mouse/human hybrid <400>SEQUENCE: 46 ctagccgcca ccatggagac agacacactc ctgctatggg tactgctgctcggcggtggt 60 acctctgtct gtgtgaggac gatacccatg acgacgagtg ggttccaggttccactggtg 120 acgaagatca ggtagatccg cggttaatca cccaaggtcc aaggtgaccactgcttctag 180 tccatctagg cgccaattag gacggtactg ccattcga 218 <210> SEQID NO 47 <211> LENGTH: 205 <212> TYPE: DNA <213> ORGANISM: hybrid <220>FEATURE: <221> NAME/KEY: misc_feature <223> OTHER INFORMATION:mouse/human hybrid <400> SEQUENCE: 47 ctagcggtac catgagatta gcagtaggcgccttattagt atgcgcagta ctccgccatg 60 gtactctaat cgtcatccgc ggaataatcatacgcgtcat gagggattat gtctcgcaga 120 agatcaggta gatccgcggt taatcgacggtaccttatac agagcgtctt ctagtccatc 180 taggcgccaa ttagctgcca ttcga 205<210> SEQ ID NO 48 <211> LENGTH: 207 <212> TYPE: DNA <213> ORGANISM:hybrid <220> FEATURE: <221> NAME/KEY: misc_feature <223> OTHERINFORMATION: mouse/human hybrid <400> SEQUENCE: 48 ctagccgcca ccatgggattagcagtaggc gccttattag tatgcgcagt cgccggtggt 60 accctaatcg tcatccgcggaataatcata cgcgtcaact cggattatgt ctcgcagaag 120 atcaggtaga tccgcggttaatcgacgtga gcctaataca gagcgtcttc tagtccatct 180 aggcgccaat tagctgcgtacattcga 207 <210> SEQ ID NO 49 <211> LENGTH: 31 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:synthetic DNA <400> SEQUENCE: 49 ggaattccac catggcgacc tccacgggtc g 31<210> SEQ ID NO 50 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: syntheticDNA <400> SEQUENCE: 50 tgaccagggt cccgtcgcg 19 <210> SEQ ID NO 51 <211>LENGTH: 39 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: synthetic DNA <400> SEQUENCE: 51gaggaccagg tggaccccag gctgatccac ggcaaggat 39 <210> SEQ ID NO 52 <211>LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:52 Glu Asp Gln Val Asp Pro Arg Leu Ile Asp Gly Lys Asp 1 5 10

1. A method of modifying lysosomal hydrolases comprising contacting saidlysosomal hydrolases with an isolated GlcNAc-phosphotransferase toproduce a modified lysosomal hydrolase.
 2. The method of claim 1,further comprising purifying said modified lysosomal hydrolase aftersaid contacting.
 3. The method of claim 1, wherein said lysosomalhydrolase comprises an asparagine-linked oligosaccharide with a highmannose structure.
 4. The method of claim 1, wherein saidGlcNAc-phosphotransferase catalyzes the transfer ofN-acetylglucosamine-1-phosphate from UDP-GlcNAc to a mannose on thehydrolase.
 5. The method of claim 1, wherein said lysosomal hydrolasesare recombinant hydrolases.
 6. The method of claim 1, wherein saidGlcNAc-phosphotransferase comprises SEQ ID NO:1, SEQ ID NO:2, and SEQ IDNO:3.
 7. The method of claim 1, wherein said GlcNAc-phosphotransferasecomprises amino acids 1-928 of SEQ ID NO:1, amino acids 1-328 of SEQ IDNO:2, and amino acids 25-305 of SEQ ID NO:3.
 8. The method of claim 1,wherein said GlcNAc-phosphotransferase comprises SEQ ID NO:15, SEQ IDNO:8, and SEQ ID NO:9.
 9. The method of claim 1, wherein said lysosomalhydrolase is selected from the group consisting of α-glucosidase,α-iduronidase, α-galactosidase A, arylsulfatase,N-acetlygalactosamine-6-sulfatase, β-galactosidase, iduronate2-sulfatase, ceramidase, galactocerebrosidase, B-glucoronidase, HeparanN-sulfatase, N-Acetyl-α-glucosaminidase, Acetyl CoA-α-glucosaminideN-acetyl transferase, N-acetyl-glucosamine-6 sulfatase, Galactose6-sulfatase, Arylsulfatase A, Arylsulfatase B, Arylsulfatase C,Arylsulfatase A Cerebroside, Ganglioside, Acid β-galactosidase G_(M1)Galglioside, Acid β-galactosidase, Hexosaminidase A, Hexosaminidase B,α-fucosidase, α-N-Acetyl galactosaminidase, Glycoprotein Neuraminidase,Aspartylglucosamine amidase, Acid Lipase, Acid Ceramidase, LysosomalSphingomyelinase, Sphingomyelinase, and Glucocerebrosidaseβ-Glucosidase.
 10. The method of claim 1, further comprising contactingsaid modified lysosomal hydrolase with an isolated phosphodiesterα-GlcNAcase.
 11. The method of claim 10, wherein said phosphodiesterα-GlcNAcase catalyzes the removal of N-acetylglucosamine from saidmodified lysosomal hydrolases and generates a terminal mannose6-phosphate on said hydrolase.
 12. The method of claim 10, wherein saidphosphodiester α-GlcNAcase comprises the amino acid sequence in SEQ IDNO:6.
 13. The method of claim 10, wherein said phosphodiesterα-GlcNAcase comprises amino acids 50-515 of SEQ ID NO:6.
 14. The methodof claim 10, wherein said phosphodiester α-GlcNAcase comprises the aminoacid sequence in SEQ ID NO:14.
 15. A highly phosphorylated purifiedlysosomal hydrolase comprising a mannose 6-phosphate.
 16. A method ofpreparing a phosphorylated lysosomal hydrolase comprising contactingsaid lysosomal hydrolase with an isolated phosphodiester α-GlcNAcase,wherein said lysosomal hydrolase comprises a terminal mannose6-phosphate.
 17. The method of claim 16, wherein said method furthercomprises purifying the phosphorylated lysosomal hydrolase.
 18. Themethod of claim 16, wherein said lysosomal hydrolase is selected fromthe group consisting of α-glucosidase, α-iduronidase, α-galactosidase A,arylsulfatase, N-acetlygalactosamine-6-sulfatase, β-galactosidase,iduronate 2-sulfatase, ceramidase, galactocerebrosidase,B-glucoronidase, Heparan N-sulfatase, N-Acetyl-α-glucosaminidase, AcetylCoA-α-glucosaminide N-acetyl transferase, N-acetyl-glucosamine-6sulfatase, Galacose 6-sulfatase, Arylsulfatase A, Arylsulfatase B,Arylsulfatase C, Arylsulfatase A Cerebroside, Ganglioside, Acidβ-galactosidase G_(M1) Galglioside, Acid β-galactosidase, HexosaminidaseA, Hexosaminidase B, α-fucosidase, α-N-Acetyl galactosaminidase,Glycoprotein Neuraminidase, Aspartylglucosamine amidase, Acid Lipase,Acid Ceramidase, Lysosomal Sphingomyelinase, Sphingomyelinase, andGlucocerebrosidase β-Glucosidase.
 19. The method of claim 16, whereinsaid phosphodiester α-GlcNAcase catalyzes the removal ofN-acetylglucosamine from said modified lysosomal hydrolases andgenerates a terminal mannose 6-phosphate on said hydrolase.
 20. Themethod of claim 16, wherein said phosphodiester α-GlcNAcase comprisesthe amino acid sequence in SEQ ID NO:6.
 21. The method of claim 16,wherein said phosphodiester α-GlcNAcase comprises amino acids 50-515 ofSEQ ID NO:6.
 22. The method of claim 16, wherein said phosphodiesterα-GlcNAcase comprises the amino acid sequence in SEQ ID NO:14.
 23. Alysosomal hydrolase obtained by the method of claim
 16. 24. A method ofpreparing a phosphorylated lysosomal hydrolase comprising: contactingsaid lysosomal hydrolase with an isolated GlcNAc-phosphotransferase toproduce a modified lysosomal hydrolase; and contacting said modifiedlysosomal hydrolase with an isolated phosphodiester α-GlcNAcase.
 25. Themethod of claim 24, further comprising purifying said phosphorylatedlysosomal hydrolase after said contacting with the isolatedphosphodiester α-GlcNAcase.
 26. The method of claim 24, furthercomprising purifying said modified lysosomal hydrolase prior to saidcontacting with the isolated phosphodiester α-GlcNAcase.
 27. Aphosphorylated lysosomal hydrolase obtained by the method of claim 24.28. The method of claim 24, wherein said GlcNAc-phosphotransferasecomprises SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.
 29. The method ofclaim 24, wherein said GlcNAc-phosphotransferase comprises amino acid1-928 of SEQ ID NO:1, amino acids 1-328 of SEQ ID NO:2, and amino acids25-305 of SEQ ID NO:3.
 30. The method of claim 24, wherein saidGlcNAc-phosphotransferase comprises SEQ ID NO:15, SEQ ID NO:8, and SEQID NO:9.
 31. The method of claim 24, wherein said phosphodiesterα-GlcNAcase comprises the amino acid SEQ ID NO:6.
 32. The method ofclaim 24, wherein said phosphodiester α-GlcNAcase comprises amino acids50-515 of SEQ ID NO:6.
 33. The method of claim 24, wherein saidlysosomal hydrolase is selected from the group consisting ofα-glucosidase, α-iduronidase, α-galactosidase A, arylsulfatase,N-acetlygalactosamine-6-sulfatase, β-galactosidase, iduronate2-sulfatase, ceramidase, galactocerebrosidase, B-glucoronidase, HeparanN-sulfatase, N-Acetyl-α-glucosaminidase, Acetyl CoA-α-glucosaminideN-acetyl transferase, N-acetyl-glucosamine-6 sulfatase, Galactose6-sulfatase, Arylsulfatase A, Arylsulfatase B, Arylsulfatase C,Arylsulfatase A Cerebroside, Ganglioside, Acid β-galactosidase G_(M1)Galglioside, Acid β-galactosidase, Hexosaminidase A, Hexosaminidase B,α-fucosidase, α-N-Acetyl galactosaminidase, Glycoprotein Neuraminidase,Aspartylglucosamine amidase, Acid Lipase, Acid Ceramidase, LysosomalSphingomyelinase, Sphingomyelinase, and Glucocerebrosidaseβ-Glucosidase.
 34. A method of producing a high mannose lysosomalhydrolase comprising: culturing transformed cells comprising arecombinant polynucleotide which encodes for a recombinant hydrolase inthe presence of an α1,2-mannosidase inhibitor; and recovering the highmannose recombinant hydrolase.
 35. The method of claim 34, wherein saidlysosomal hydrolase is selected from the group consisting ofα-glucosidase, α-iduronidase, α-galactosidase A, arylsulfatase,N-acetlygalactosamine-6-sulfatase, β-galactosidase, iduronate2-sulfatase, ceramidase, galactocerebrosidase, B-glucoronidase, HeparanN-sulfatase, N-Acetyl-α-glucosaminidase, Acetyl CoA-α-glucosaminideN-acetyl transferase, N-acetyl-glucosamine-6 sulfatase, Galactose6-sulfatase, Arylsulfatase A, Arylsulfatase B, Arylsulfatase C,Arylsulfatase A Cerebroside, Ganglioside, Acid β-galactosidase G_(M1)Galglioside, Acid β-galactosidase, Hexosaminidase A, Hexosaminidase B,α-fucosidase, α-N-Acetyl galactosaminidase, Glycoprotein Neuraminidase,Aspartylglucosamine amidase, Acid Lipase, Acid Ceramidase, LysosomalSphingomyelinase, Sphingomyelinase, and Glucocerebrosidaseβ-Glucosidase.
 36. The method of claim 34, wherein said lysosomalhydrolase is α-glucosidase.
 37. The method of claim 34, wherein thealpha 1,2-mannosidase inhibitor is selected from the group consisting ofdeoxymannojirimycin (dMM), kifunensine, D-Mannonolactam amidrazone, andN-butyl-deoxymannojirimycin.
 38. The method of claim 34, wherein thealpha 1,2-mannosidase inhibitor is kifunensine.
 39. A high mannoselysosomal hydrolase prepared according to the method of claim 34.